Stabilized liquid and lyophilized adamts13 formulations

ABSTRACT

The present invention relates to formulations of ADAMTS13 with enhanced or desirable properties. As such, the invention provides liquid and lyophilized formulations of ADAMTS13 that are suitable for pharmaceutical administration. Among other aspects, the present invention also provides methods of treating various diseases and conditions related to VWF and/or ADAMTS13 dysfunction in a subject. Also provided herein are kits comprising ADAMTS13 formulations useful for the treatment of various diseases and conditions.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.14/088,169, which is a continuation of U.S. application Ser. No.12/887,424, filed Sep. 21, 2010, which claims the benefit of U.S.Provisional Application No. 61/244,353, filed Sep. 21, 2009, which areexpressly incorporated herein by reference in their entireties for allpurposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

NOT APPLICABLE

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

BACKGROUND OF THE INVENTION

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin typeI motifs) proteins are a family of metalloproteinases containing numberof conserved domains, including a zinc-dependant catalytic domain, acysteine-rich domain, a disintegrin-like domain, and at least one, andin most cases multiple, thrombospondin type I repeats (for review, seeNicholson et al., BMC Evol Biol. 2005 Feb. 4; 5(1):11). These proteins,which are evolutionarily related to the ADAM and MMP families ofmetalloproteinases (Jones G C, Curr Pharm Biotechnol. 2006 February;7(1):25-31), are secreted enzymes that have been linked to a number ofdiseases and conditions including thrombotic thrombocytopenic purpura(TTP) (Moake J L, Semin Hematol. 2004 January; 41(1):4-14), connectivetissue disorders, cancers, inflammation (Nicholson et al.), and severeplasmodium falciparum malaria (Larkin et al., PLoS Pathog. 2009 March;5(3):e1000349). Because of these associations, the ADAMTS enzymes havebeen recognized as potential therapeutic targets for a number ofpathologies (Jones G C, Curr Pharm Biotechnol. 2006 February;7(1):25-31).

One ADAMTS family member, ADAMTS13, cleaves von Willebrand factor (vWF)between residues Tyr 1605 and Met 1606. Loss of ADAMTS13 activity hasbeen linked to a number of conditions, such as TTP (Moake J L, SeminHematol. 2004 January; 41(1):4-14), acute and chronic inflammation(Chauhan et al., J Exp Med. 2008 Sep. 1; 205(9):2065-74), and mostrecently, severe plasmodium falciparum malaria (Larkin et al., PLoSPathog. 2009 March; 5(3): e1000349).

Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized bythrombotic microangiopathy, thrombocytopenia and microvascularthrombosis that can cause various degrees of tissue ischemia andinfarction. Clinically, TTP patients are diagnosed by symptoms such asthrombocytopenia, schistocytes (fragments of erythrocytes) and elevatedlevels of lactate dehydrogenase (Moake J L. Thromboticmicroangiopathies. N Engl J Med. 2002; 347:589-600; Moake J L. vonWillebrand factor, ADAMTS-13, and thrombotic thrombocytopenic purpura.Semin Hematol. 2004; 41:4-14; Sadler J E, Moake J L, Miyata T, George JN. Recent advances in thrombotic thrombocytopenic purpura. Hematology(Am Soc Hematol Educ Program). 2004: 407-423; Sadler J E. New conceptsin von Willebrand disease. Annu Rev Med. 2005; 56:173-191).

In 1982, Moake et al. found unusually large von Willebrand factor(UL-vWF) multimers in the plasma of the patients with chronic relapsingTTP (Moake J L, Rudy C K, Troll J H, Weinstein M J, Colannino N M,Azocar J, Seder R H, Hong S L, Deykin D. Unusually large plasma factorVIII:von Willebrand factor multimers in chronic relapsing thromboticthrombocytopenic purpura. N Engl J Med. 1982; 307:1432-1435). The linkbetween UL-vWF and TTP gained support with independent findings byFurlan et al. and Tsai and Lian that most patients suffering from TTPare deficient in a plasma metalloprotease, now known to be ADAMTS13,that cleaves vWF (Furlan M, Robles R, Solenthaler M, Wassmer M, SandozP, Laemmle B. Deficient activity of von Willebrand factor-cleavingprotease in chronic relapsing thrombotic thrombocytopenic purpura.Blood. 1997; 89:3097-3103; Tsai H M, Sussman, I I, Ginsburg D, LankhofH, Sixma J J, Nagel R L. Proteolytic cleavage of recombinant type 2A vonWillebrand factor mutants R834W and R834Q: inhibition by doxycycline andby monoclonal antibody VP-1. Blood. 1997; 89:1954-1962; Tsai H M, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acutethrombotic thrombocytopenic purpura. N Engl J Med. 1998; 339:1585-1594).

The ADAMTS13 protease is a 190 kDa glycosylated protein producedpredominantly by the liver (Levy G G, Nichols W C, Lian E C, Foroud T,McClintick J N, McGee B M, Yang A Y, Siemieniak D R, Stark K R, GruppoR, Sarode R, Shurin S B, Chandrasekaran V, Stabler S P, Sabio H,Bouhassira E E, Upshaw J D, Jr., Ginsburg D, Tsai H M. Mutations in amember of the ADAMTS gene family cause thrombotic thrombocytopenicpurpura. Nature. 2001; 413:488-494; Fujikawa K, Suzuki H, McMullen B,Chung D. Purification of human von Willebrand factor-cleaving proteaseand its identification as a new member of the metalloproteinase family.Blood. 2001; 98:1662-1666; Zheng X, Chung D, Takayama T K, Majerus E M,Sadler J E, Fujikawa K. Structure of von Willebrand factor-cleavingprotease (ADAMTS13), a metalloprotease involved in thromboticthrombocytopenic purpura. J Biol Chem. 2001; 276:41059-41063; Soejima K,Mimura N, Hirashima M, Maeda H, Hamamoto T, Nakagaki T, Nozaki C. Anovel human metalloprotease synthesized in the liver and secreted intothe blood: possibly, the von Willebrand factor-cleaving protease; JBiochem (Tokyo). 2001; 130:475-480; Gerritsen H E, Robles R, Lammle B,Furlan M. Partial amino acid sequence of purified von Willebrandfactor-cleaving protease. Blood. 2001; 98:1654-1661).

Mutations in the ADAMTS13 gene have been shown to cause TTP (Levy G G,Nichols W C, Lian E C, Foroud T, McClintick J N, McGee B M, Yang A Y,Siemieniak D R, Stark K R, Gruppo R, Sarode R, Shurin S B,Chandrasekaran V, Stabler S P, Sabio H, Bouhassira E E, Upshaw J D, Jr.,Ginsburg D, Tsai H M. Mutations in a member of the ADAMTS gene familycause thrombotic thrombocytopenic purpura. Nature. 2001; 413:488-494).Idiopathic TTP, often caused by autoantibodies inhibiting ADAMTS-13activity, is a more common disorder that occurs in adults and olderchildren and can recur at regular intervals in 11-36% of patients (TsaiH M, Lian E C. Antibodies to von Willebrand factor-cleaving protease inacute thrombotic thrombocytopenic purpura. N Engl J Med. 1998;339:1585-1594; Furlan M, Lammle B. Deficiency of von Willebrandfactor-cleaving protease in familial and acquired thromboticthrombocytopenic purpura. Baillieres Clin Haematol. 1998; 11:509-514).

Non neutralizing autoantibodies could also inhibit ADAMTS activity byinducing clearance from circulation (Scheiflinger F, Knobl P, TrattnerB, Plaimauer B, Mohr G, Dockal M, Dorner F, Rieger M. NonneutralizingIgM and IgG antibodies to von Willebrand factor-cleaving protease(ADAMTS-13) in a patient with thrombotic thrombocytopenic purpura.Blood. 2003; 102:3241-3243). Plasma ADAMTS13 activity in healthy adultsranges from 50% to 178% (Moake J L. Thrombotic thrombocytopenic purpuraand the hemolytic uremic syndrome. Arch Pathol Lab Med. 2002;126:1430-1433). In most patients with familial or acquired TTP, plasmaADAMTS13 activity is absent or less than 5% of the normal. Withouttreatment the mortality rate exceeds 90%, but plasma therapy has reducedmortality to about 20% (Moake J L. Thrombotic thrombocytopenic purpuraand the hemolytic uremic syndrome. Arch Pathol Lab Med. 2002;126:1430-1433).

vWF synthesized in megakaryocytes and endothelial cells is stored inplatelet—granules and Weibel-Palade bodies, respectively, as ultra largevWF (UL-vWF) (Moake J L, Rudy C K, Troll J H, Weinstein M J, Colannino NM, Azocar J, Seder R H, Hong S L, Deykin D. Unusually large plasmafactor VIII:von Willebrand factor multimers in chronic relapsingthrombotic thrombocytopenic purpura. N Engl J Med. 1982; 307:1432-1435;Wagner D D, Olmsted J B, Marder V J. Immunolocalization of vonWillebrand protein in Weibel-Palade bodies of human endothelial cells. JCell Biol. 1982; 95:355-360; Wagner D D, Bonfanti R. von Willebrandfactor and the endothelium. Mayo Clin Proc. 1991; 66:621-627; Sporn L A,Marder V J, Wagner D D. von Willebrand factor released fromWeibel-Palade bodies binds more avidly to extracellular matrix than thatsecreted constitutively. Blood. 1987; 69:1531-1534; Tsai H M, Nagel R L,Hatcher V B, Sussman, I I. Endothelial cell-derived high molecularweight von Willebrand factor is converted into the plasma multimerpattern by granulocyte proteases. Biochem Biophys Res Commun. 1989;158:980-985; Tsai H M, Nagel R L, Hatcher V B, Sussman, I I. Multimericcomposition of endothelial cell-derived von Willebrand factor. Blood.1989; 73:2074-2076). Once secreted from endothelial cells, these UL-vWFmultimers are cleaved by ADAMTS13 in circulation into a series ofsmaller multimers at specific cleavage sites within the vWF molecule(Tsai H M, Nagel R L, Hatcher V B, Sussman, I I. Endothelialcell-derived high molecular weight von Willebrand factor is convertedinto the plasma multimer pattern by granulocyte proteases. BiochemBiophys Res Commun. 1989; 158:980-985; Dent J A, Galbusera M, Ruggeri ZM. Heterogeneity of plasma von Willebrand factor multimers resultingfrom proteolysis of the constituent subunit. J Clin Invest. 1991;88:774-782; Furlan M, Robles R, Affolter D, Meyer D, Baillod P, LammleB. Triplet structure of von Willebrand factor reflects proteolyticdegradation of high molecular weight multimers. Proc Natl Acad Sci USA.1993; 90:7503-7507).

ADAMTS13 cleaves at the Tyr842-Met843 bond in the central A2 domain ofthe mature vWF subunit and requires zinc or calcium for activity (Dent JA, Berkowitz S D, Ware J, Kasper C K, Ruggeri Z M. Identification of acleavage site directing the immunochemical detection of molecularabnormalities in type IIA von Willebrand factor. Proc Natl Acad Sci USA.1990; 87:6306-6310). vWF exists in “ball-of-yarn” and filamentous formas seen by electron microscopy (Slayter H, Loscalzo J, Bockenstedt P,Handin R I. Native conformation of human von Willebrand protein.Analysis by electron microscopy and quasi-elastic light scattering. JBiol. Chem. 1985; 260:8559-8563). Furthermore, atomic force microscopyconfirms that vWF exits in a globular conformation under staticconditions and an unfolded filamentous state after exposure to shearstress (Siedlecki C A, Lestini B J, Kottke-Marchant K K, Eppell S J,Wilson D L, Marchant R E. Shear-dependent changes in thethree-dimensional structure of human von Willebrand factor. Blood. 1996;88:2939-2950). This could occur also in vivo when one end of the vWFfilament is anchored to a surface.

Thrombi of TTP patients consist of little fibrin and mainly of vWF andplatelets, suggesting vWF-mediated platelet aggregation as a cause ofthrombosis (Asada Y, Sumiyoshi A, Hayashi T, Suzumiya J, Kaketani K.Immunohistochemistry of vascular lesion in thrombotic thrombocytopenicpurpura, with special reference to factor VIII related antigen. ThrombRes. 1985; 38:469-479). Patients with relapsing TTP have ultra-largemultimers in the plasma. The UL-vWF multimers accumulate over timebecause the persistence of the inhibitor (Anti-ADAMTS13 Ab) decreasesADAMTS13 activity. The UL-vWF multimers are hyperactive and unfold as aresult of shear stress causing platelet aggregation, resulting inintravascular thrombosis (Tsai H M. Von Willebrand factor, ADAMTS13, andthrombotic thrombocytopenic purpura. J Mol Med. 2002; 80:639-647; Tsai HM. Deficiency of ADAMTS-13 in thrombotic and thrombocytopenic purpura. JThromb Haemost. 2003; 1:2038-2040; discussion 2040-2035).

It is believed that the presence of hyper-reactive UL-vWF multimers inthe plasma due to ADAMTS13 deficiency could be associated with anincreased risk of arterial thrombosis linked to coronary heart disease.

Accordingly, there is a need in the art for pharmaceutical formulationsof ADAMTS13 proteins suitable for the treatment of various diseases andconditions associated with ADAMTS13 and VWF dysfunction. The presentinvention provides, among other aspects, ADAMTS13 formulations suitablefor pharmaceutical administration, as well as methods of treatingsubjects with diseases or conditions associated with ADAMTS13 and VWFdysfunction.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides formulations of ADAMTS13(A13) suitable for pharmaceutical administration. Advantageously, insome embodiments, the invention provides A13 formulations that may bestored for extended periods of time without loosing activity or becomingoverly aggregated. In certain embodiments, the formulations of theinvention may be stored for at least 6 months at temperatures up to atleast about 37° C.

In one aspect, the present invention provides a stabilized formulationof ADAMTS13 (A13) comprising (a) 0.05 mg/ml to 10.0 mg/ml ADAMTS13; (b)0 mM to 200 mM of a pharmaceutically acceptable salt; (c) 0.5 mM to 20mM calcium; (d) a sugar and/or sugar alcohol; (e) a nonionic surfactant;and (f) a buffering agent for maintaining a pH between 6.0 and 8.0.

In a related aspect, the present invention provides a stabilizedlyophilized formulation of ADAMTS13 (A13), wherein the formulation islyophilized from a liquid formulation comprising (a) 0.05 mg/ml to 10.0mg/ml ADAMTS13; (b) 0 mM to 200 mM of a pharmaceutically acceptablesalt; (c) 0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e)a nonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0.

In one aspect of the invention, recombinant ADAMTS13 (rA13) formulationssuitable for pharmaceutical administration are provided. In someembodiments, the formulations provided herein have high specificactivities and are stable upon storage for extended periods of time.

In another aspect of the invention, formulations are provided that havereduced dimerization or aggregation of A13 and rA13. In certainembodiments, the formulations provided herein retard the formation ofA13 and rA13 dimers and aggregation when stored for extended periods oftime. In some embodiments, these formulations are suitable forpharmaceutical administration.

In one aspect, the present invention relates to a method of treating orpreventing a disorder associated with the formation and/or presence ofone or more thrombus and to a method of disintegrating one or morethrombus in a patient in need thereof. Examples of disorders associatedwith the formation and/or the presence of one or more thrombus arehereditary thrombotic thrombocytopenic purpura (TTP), acquired TTP,arterial thrombosis, acute myocardial infarction (AMI), stroke, sepsis,and disseminated intravascular coagulation (DIC). In one embodiment, themethods of treating or preventing include the administration of an A13or rA13 formulation provided herein.

In another aspect, the present invention provides methods of treating orpreventing an infarction in a patient in need thereof. In certainembodiments, methods are provided for treating or preventing a diseaseor condition associated with an infarction, including withoutlimitation, myocardial infarction (heart attack), pulmonary embolism,cerebrovascular events such as stroke, peripheral artery occlusivedisease (such as gangrene), antiphospholipid syndrome, sepsis,giant-cell arteritis (GCA), hernia, and volvulus. In one embodiment, themethods of treating or preventing include the administration of an A13or rA13 formulation provided herein.

In one aspect, the present invention provides methods of formulating A13or rA13 with high stability and/or high specific activities. In certainembodiments, the methods are useful for formulating solutions andlyophilisates that can be stored for extended periods of time attemperatures up to at least about 37° C. without loosing high specificactivities desirable for pharmaceutical administration.

In another aspect, kits for the pharmaceutical administration of A13 orrA13 are provided herein. The kits of the invention, in certainembodiments, may comprise liquid or lyophilized formulations that may bestored for extended periods of time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B. Recombinant ADAMTS13 was formulated in buffercontaining 150 mM NaCl, 2% sucrose, 0.05% polysorbate 80, at a pH of 7.0with 20 mM of a buffering agent selected from (1) histidine, (2)phosphate buffer, or (3) sodium citrate. Solutions were stored at 4° C.or 37° C. for up to 6 months. (A) FRETS-VWF73 activity and (B) ADAMTS13protein concentration as measured by ELISA, were determined at theindicated time points.

FIG. 2A and FIG. 2B. Recombinant ADAMTS13 was formulated in buffercontaining 150 mM NaCl, 2% sucrose, 0.05% polysorbate 80, at a pH of 7.0with 20 mM of a buffering agent selected from (1) histidine, (2)phosphate buffer, or (3) sodium citrate and lyophilized. Lyophilizedformulations were stored at 4° C. or 37° C. for up to 6 months. ADAMTS13formulations were reconstituted with sterile water and (A) FRETS-VWF73activity and (B) ADAMTS13 protein concentration as measured by ELISA,were determined at the indicated time points.

FIG. 3. Recombinant ADAMTS13 was formulated in buffer containing 150 mMNaCl, 2% sucrose, 0.05% polysorbate 80, at a pH of 7.0 with 20 mM of abuffering agent selected from (1) histidine, (2) phosphate buffer, or(3) sodium citrate and samples were either loaded directly onto aSuperose 6 gel filtration column or lyophilized and reconstituted withsterile water prior to loading. The ADAMTS13 formulations were thenanalyzed by gel filtration.

FIGS. 4A and 4B. Recombinant ADAMTS13 was formulated in buffercontaining 150 mM NaCl, 2% sucrose, 0.05% polysorbate 80, and 20 mMhistidine at pH 7.0. Solutions were stored at (A) 4° C. or (B) 37° C.for up to 6 months. ADAMTS13 formulations were then analyzed by gelfiltration at the indicated time points.

FIGS. 5A and 5B. Recombinant ADAMTS13 was formulated in buffercontaining 150 mM NaCl, 2% sucrose, 0.05% polysorbate 80, and 20 mMhistidine at pH 7.0 and then lyophilized. Lyophilized formulations werethen stored at (A) 4° C. or (B) 37° C. for up to 6 months. ADAMTS13formulations were reconstituted in sterile water at the indicated timepoints and analyzed by gel filtration.

FIGS. 6A and 6B. Recombinant ADAMTS13 was formulated in buffercontaining 150 mM NaCl, 2% sucrose, 0.05% polysorbate 80, and 20 mMhistidine at pH 7.0 and then stored (A) in solution or (B) lyophilizedat 4° C. or 37° C. for up to 6 months. Lyophilized formulations werereconstituted in sterile water and samples stored in solution andlyophilized formulations were characterized by dynamic light scatteringanalysis.

FIGS. 7A and 7B. Recombinant ADAMTS13 was formulated in buffercontaining 150 mM NaCl, 2% sucrose, 0.05% polysorbate 80, and 20 mMsodium phosphate buffer at pH 7.0 and then stored (A) in solution or (B)lyophilized at 4° C. or 37° C. for up to 6 months. Lyophilizedformulations were reconstituted in sterile water and samples stored insolution and lyophilized formulations were characterized by dynamiclight scattering analysis.

FIGS. 8A and 8B. Recombinant ADAMTS13 was formulated in buffercontaining 150 mM NaCl, 2% sucrose, 0.05% polysorbate 80, and 20 mMtri-sodium citrate at pH 7.0 and then stored (A) in solution or (B)lyophilized at 4° C. or 37° C. for up to 6 months. Lyophilizedformulations were reconstituted in sterile water and samples stored insolution and lyophilized formulations were characterized by dynamiclight scattering analysis.

FIG. 9. Recombinant ADAMTS13 was formulated in buffer containing 150 mMNaCl, 2% sucrose, 0.05% polysorbate 80, and 20 mM histidine at pH 7.0and then lyophilized. Lyophilized samples were stored at 4° C. or 37° C.for 3 months, and then reconstituted in sterile water. ADAMTS13 sampleswere then characterized by fourier transform infrared spectroscopy andthe results were compared to freshly formulated and lyophilized sample.

FIG. 10. Recombinant ADAMTS13 was formulated in buffer containing 150 mMNaCl, 2% sucrose, 0.05% polysorbate 80, at a pH of 7.0 with 20 mM of abuffering agent selected from (1) histidine, (2) phosphate buffer, or(3) sodium citrate. Solutions were stored at 4° C. or 37° C. for 6months. Lyophilized formulations were reconstituted with sterile waterand the purity of all samples were determined by reverse phase-HPLCanalysis.

FIG. 11. Recombinant ADAMTS13 was formulated in buffer containing 150 mMNaCl, 2% sucrose, 0.05% polysorbate 80, at a pH of 7.0 with 20 mM of abuffering agent selected from (1) histidine, (2) phosphate buffer, or(3) sodium citrate. Samples of the solution formulations were alsolyophilized and reconstituted with sterile water. Solution andreconstituted lyophilized formulations were characterized bydifferential scanning calorimetry analysis.

FIG. 12. Recombinant ADAMTS13 was formulated in buffer containing 150 mMNaCl, 2% sucrose, 0.05% polysorbate 80, at a pH of 7.0 with 20 mM of abuffering agent selected from (1) histidine, (2) phosphate buffer, or(3) sodium citrate. The z-average diameter of the ADAMTS13 in theformulations was characterized by dynamic light scattering analysis.

FIGS. 13A and 13B. Recombinant ADAMTS13 was formulated in buffercontaining 150 mM NaCl, 0%-2% sucrose, 0%-3% mannitol, 0 mM-2 mMcalcium, 0.05% polysorbate 80, and 20 mM histidine at pH 7.0 andanalyzed by gel filtration. The full chromatograph is shown in panel(A), a zoomed section of the dimer-peak is shown in panel (B).

FIG. 14. Recombinant ADAMTS13 was formulated in buffer containing 150 mMNaCl and 20 mM histidine at pH 5.5, 6.5, 7.5, 8.5, and 9.5. Formulationswere stored at 4° C. or 40° C. for 24 hours. FRETS-VWF73 activity andADAMTS13 protein concentration as measured by ELISA, were thendetermined for the indicated pH formulations.

FIG. 15. Recombinant ADAMTS13 was formulated in buffer containing 150 mMNaCl, 2% sucrose, 0.05% polysorbate 80, and 20 mM histidine at pH 7.0with (12B and D) and without (12A and C) 2 mM CaCl₂. The liquidformulations were stored at either 25° C. (12A and B) or 37° C. (12C andD) for three weeks. Enzyme stability was measured by FRETS-VWF73 assayat the indicated time points.

FIGS. 16A and 16B. (A) Recombinant ADAMTS13 was loaded onto a Superose 6GL gel filtration column equilibrated with a buffer containing 20 mMNa₂HPO₄ (pH 7.5) and 500 mM NaCl, and molecular species were separatedby size and shape. Three elution pools were created corresponding toaggregated A13 (Pool 1), dimeric A13 (Pool 2), and monomeric A13 (Pool3). The FRETS-VWF73 activity of each pool was then determined. (B) Theoligomeric state and polydispersity of the pooled fractions was furtherassessed by dynamic light scattering analysis.

FIG. 17A-17F. Recombinant ADAMTS13 was formulated in buffer containing2% sucrose, 0.05% polysorbate 80, 20 mM histidine at pH 7.0, and NaCl at(A) 0 mM, (B) 30 mM, (C) 60 mM, (D) 90 mM, (E) 120 mM, or (F) 150 mM.The formulated ADAMTS13 compositions were then lyophilized and visuallyinspected for the appearance of the resulting lyocake.

FIG. 18. Recombinant ADAMTS13 lyocakes produced from formulationscontaining from 0 to 150 mM NaCl were reconstituted in water. Theaggregation states of the reconstituted ADAMTS13 proteins weredetermined by gel filtration analysis.

FIG. 19. Recombinant ADAMTS13 was formulated in buffer containing 0.05%polysorbate 80, 20 mM histidine at pH 7.0, sucrose at a concentrationfrom 5 mM to 20 mM, and NaCl at a concentration from 0 to 150 mM. Theactivity of the ADAMTS13 protein in each formulation was determined byFRETS-VWF73 assay.

FIGS. 20A and 20B. High salt and a low salt buffers were lyophilized.The high salt buffer (A) contained 20 mM histidine (pH 7.0), 150 mMNaCl, 2% sucrose, 0.05% polysorbate 80, and 2 mM CaCl₂, while the lowsalt buffer (B) contained 20 mM histidine (pH 7.0), 30 mM NaCl, 1%sucrose, 3% mannitol, 0.05% polysorbate 80, and 2 mM CaCl₂. The twoformulations were then lyophilized under standard conditions and theresulting lyocakes were then visually inspected.

FIGS. 21A and 21B. The multimeric state of recombinant ADAMTS13 isolatedby cation exchange chromatography (A) before and (B) after gelfiltration was analyzed by size exclusion chromatography using aSuperose 6 GL column.

FIGS. 22A and 22B. The multimeric state of recombinant ADAMTS13 isolatedby cation exchange chromatography (A) before and (B) after gelfiltration was analyzed by dynamic light scattering.

FIGS. 23A and 23B. (A) Standard and (B) extended protocols forlyophilization of rADAMTS13 formulations.

FIGS. 24A and 24D. Comparison of rADAMTS13 lyocakes produced bylyophilization of (A) rA13 formulations 1 to 4 using a standardlyophilization protocol, (B) rA13 formulations 1 to 4 using an extendedlyophilization protocol, (C) rA13 formulations 5 to 8 using a standardlyophilization protocol, and (D) rA13 formulations 5 to 8 using anextended lyophilization protocol. Formulations and lyophilizationprotocols used for the generation of this data can be found in example12.

FIGS. 25A and 25B. The oligomeric state and polydispersity of ADAMTS13compositions formulated with (A) 30 mM NaCl or (B) 60 mM NaCl wasassessed by dynamic light scattering analysis after lyophilization witha standard or extended protocol.

FIG. 26. Domain structure of human von Willebrand factor (VWF)-cleavingprotease ADAMTS13. The precursor ADAMTS13 polypeptide consists of asignal peptide (S), a propeptide (P), followed by the structuralfeatures of the mature polypeptide comprising a catalyticmetalloprotease domain, a disintegrinlike domain, a thrombospondin type1 (TSP1) motif, a cysteine-rich and spacer domain, 7 additional TSP1repeats and 2 CUB domains. Ten potential N-glycosylation sites aremarked by closed circles.

FIG. 27. Hydrodynamic diameters of ADAMTS13 compositions formulated withdifferent buffering agents as determined by dynamic light scatteringanalysis (DLS).

FIG. 28. Influence of shear stress on recombinant human ADAMTS13 asdetermined by dynamic light scattering analysis (DLS).

FIG. 29. Freeze-thaw stability of recombinant human ADAMTS13 asdetermined by FRETS activity.

FIGS. 30A and 30B. Freeze-thaw stability of recombinant human ADAMTS13as determined by dynamic light scattering analysis (DLS).

FIG. 31. Photostability of lyophilized recombinant human ADAMTS13 asdetermined by SE-HPLC.

FIGS. 32A and 32B. Photostability of liquid and lyophilized recombinanthuman ADAMTS13 as determined by dynamic light scattering analysis (DLS).

FIG. 33. Photostability of liquid and lyophilized recombinant humanADAMTS13 as determined by FRETS activity.

FIG. 34. Influence of calcium and zinc on the FRETS activity ofrecombinant human ADAMTS13.

FIG. 35. Influence of salt and sugar content on the oligomeric state oflyophilized recombinant human ADAMTS13 formulation as determined bySE-HPLC. The sugar content of each formulation should be reported ing/L, rather than molar concentrations (e.g., 20 mM=20 g/L).

FIG. 36. Lyocakes produced by lyophilization of recombinant humanADAMTS13 formulated according to Table 15.

FIG. 37A-37C. Dynamic light scattering (DLS) analysis of the oligomericstate of recombinant human ADAMTS13 lyophilized with a standard 3 daylyophilization program, stored for 6 months at (Top Panel) 4° C.;(Middle Panel) 30° C.; and (Bottom Panel) 40° C.

FIG. 38A-38C. Dynamic light scattering (DLS) analysis of the oligomericstate of recombinant human ADAMTS13 lyophilized with an extended 10 daylyophilization program, stored for 6 months at (Top Panel) 4° C.;(Middle Panel) 30° C.; and (Bottom Panel) 40° C.

FIG. 39. Dynamic light scattering (DLS) analysis of the oligomeric stateof recombinant human ADAMTS13 formulated at different proteinconcentrations after storage at 4° C., 30° C., and 40° C.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

ADAMTS13 is a plasma metalloprotease which cleaves von Willebrand factor(VWF) multimers and down regulates their activity in plateletaggregation. Thrombotic thrombocytopenic purpura (TTP) is a severedisease associated with unusually large, hemostatically hyperactive vonWillebrand factor (VWF) and severe deficiency in ADAMTS-13. Arecombinant ADAMTS13 product candidate has been developed as arecombinant protein substitution therapy for the treatment of ADAMTS13deficiency caused TTP (Plaimauer and Scheiflinger, Semin Hematol. 2004January; 41(1):24-33; Plaimauer B et al., F. Blood. 2002 Nov. 15;100(10):3626-32. Epub 2002 Jul. 12; and Bruno K et al., J ThrombHaemost. 2005 May; 3(5):1064-73, the disclosures of which are allexpressly incorporated by reference herein in their entireties for allpurposes.)

The present invention is based in part on the discovery of liquid andlyophilized formulations of purified ADAMTS proteins with increasedstability that meet the requirements set forth by the InternationalConference on Harmonisation of Technical Requirements of Pharmaceuticalsfor Human Use (ICH Guideline, PHARMACEUTICAL DEVELOPMENT Q8(R2), 2005).

The hemostatic activity of VWF is highly dependent on the size of themultimers. The plasma metalloprotease ADAMTS13 is the main proteaseidentified that physiologically regulates the multimeric size of VWF.ADAMTS13 cleaves between Tyr 1605 and Met 1606 in the A2 domain of VWFyielding the typical cleavage fragments of 176 kDa and 140 kDa andsmaller VWF multimers found in the circulation. The human ADAMTS13 genecontains 29 exons and spans approximately 37 kb on chromosome 9q34. The4.7 kb transcript is predominantly synthesized in hepatic stellatecells, but also in vascular endothelial cells and platelets and encodesa primary translation product of 1427 amino acid residues. The precursorADAMTS13 polypeptide consists of a signal peptide and a propeptide thatC-terminally ends in a potential furin site for cleavage, followed bythe sequence of the mature VWF-cleaving protease. The mature ADAMTS13polypeptide (1353 amino acid residues) comprises the structural featurescharacteristic of all ADAMTS family members: a reprolysin-likemetalloprotease domain, a disintegrin-like domain, a centralthrombospondin type 1 (TSP1) repeat, a cysteine-rich domain harboring aRGD motif possibly important for integrin interactions, and a spacerdomain, thereafter followed by an unique combination of 7 consecutiveTSP1 repeats (TSP1/#2-8) and two CUB domains (FIG. 26).

The mature ADAMTS13 has a calculated molecular mass of about 145 kDawhereas purified plasma-derived ADAMTS13 has an apparent molecular massof about 180 kDa probably due to post-translational modificationsconsisting with present consensus sequences for 10 potentialN-glycosylation sites, and several O-glycosylation sites and oneC-mannosylation site in the TSP1 repeats. The VWF-proteolytic activityof ADAMTS13 is highly dependent on divalent cations. The active sitemotif in the metalloprotease domain contains the highly conservedHEXXHXXGXXHD motif with three histidine residues that coordinate acatalytic Zn2+ ion and a predicted Calcium binding site proposed to becoordinated by Glu 83, Asp 173, Cys 281 and Asp 284. The functionalroles of the ADAMTS13 domains have been studied mainly using in vitroassay systems, showing that the N-terminal regions from themetalloprotease to the spacer domain are crucial for VWF-cleavage.C-terminal TSP1 repeats and the CUB domains seem to be important for VWFsubstrate recognition and binding to potential surface receptors likeCD36 on endothelial cells.

One major output of protein formulation development is theidentification of inactivation pathways and aggregation or degradationpathways of the respective protein. Complete time-dependent inactivationof recombinant human ADAMTS13 protein within 1 week was observed at 40°C. in liquid formulation by FRETS activity assay. As disclosed herein,it was found that this inactivation could be reduced in the presence of2 mM Ca²⁺. At 25° C., FRETS activity did not decrease anymore in thepresence of Ca²⁺. At 4° C., liquid formulations of ADAMTS13 activitywere stable for 24 weeks in the absence of Ca²⁺.

Further, it was found that in identical lyophilized formulations thatonly slight or no inactivation occurred when the formulation was storedat 40° C. in the absence of Ca²⁺. The addition of 2 mM or 4 mM Ca²⁺ didnot influence stability at 40° C.

Accordingly, the inventors have found that time-dependent aggregation ofADAMTS13 proteins in liquid formulation (e.g., stored at 40° C.) is amajor contributor to the degradation and/or loss of enzymatic activity.Importantly, as provided by the present invention, this aggregation canbe substantially reduced by lyophilization and/or by the addition ofCa²⁺ to liquid formulations. Additionally, it has been found that theaddition of a non-ionic surfactant (e.g., 0.05% Tween 80) reduces theformation of ADAMTS13 aggregates in lyophilized formulations.Furthermore, it was found that the presence of one or more sugars and/orsugar alcohols significantly stabilized ADAMTS13 during lyophilizationand assists in the formation of an improved lyocake.

II. Definitions

As used herein, “ADAMTS13” or “A13” refer to a metalloprotease of theADAMTS (a disintegrin and metalloproteinase with thrombospondin type Imotifs) family that cleaves von Willebrand factor (vWF) between residuesTyr 1605 and Met 1606. In the context of the present invention, anADAMTS13 protein embraces any ADAMTS13 protein, for example, ADAMTS13from a mammal such as a primate, human (NP_620594), monkey, rabbit, pig,bovine (XP_610784), rodent, mouse (NP_001001322), rat (XP_342396),hamster, gerbil, canine, feline, frog (NP_001083331), chicken(XP_415435), and biologically active derivatives thereof. Mutant andvariant ADAMTS13 proteins having activity are also embraced, as arefunctional fragments and fusion proteins of the ADAMTS13 proteins.Furthermore, the ADAMTS13 proteins of the invention may further comprisetags that facilitate purification, detection, or both. The ADAMTS13proteins described herein may further be modified with a therapeuticmoiety or a moiety suitable imaging in vitro or in vivo.

Human ADAMTS13 proteins include, without limitation, polypeptidescomprising the amino acid sequence of GenBank accession number NP_620594or a processed polypeptide thereof, for example a polypeptide in whichthe signal peptide (amino acids 1 to 29) and/or propeptide (amino acids30-74) have been removed. Many natural variants of human ADAMTS13 areknown in the art, and are embraced by the formulations of the presentinvention, some of which include mutations selected from R7W, V88M,H96D, R102C, R193W, T196I, H234Q, A250V, R268P, W390C, R398H, Q448E,Q456H, P457L, P475S, C508Y, R528G, P618A, R625H, I673F, R692C, A732V,E740K, A900V, S903L, C908Y, C951G, G982R, C1024G, A1033T, R1095W,R1095W, R1123C, C1213Y, T12261, G1239V, and R1336W. Additionally,ADAMTS13 proteins include natural and recombinant proteins that havebeen mutated, for example, by one or more conservative mutations at anon-essential amino acid. Preferably, amino acids essential to theenzymatic activity of ADAMTS13 will not be mutated. These include, forexample, residues known or presumed to be essential for metal bindingsuch as residues 83, 173, 224, 228, 234, 281, and 284, and residuesfound in the active site of the enzyme, e.g., residue 225. Similarly, inthe context of the present invention, ADAMTS13 proteins includealternate isoforms, for example, isoforms lacking amino acids 275 to 305and/or 1135 to 1190 of the full-length human protein.

As used herein, the term “biologically active derivative” refers to anypolypeptide with substantially the same biological function as ADAMTS13.The polypeptide sequences of the biologically active derivatives maycomprise deletions, additions and/or substitution of one or more aminoacids whose absence, presence and/or substitution, respectively, do nothave any substantial negative impact on the biological activity ofpolypeptide. The biological activity of said polypeptides may bemeasured, for example, by the reduction or delay of platelet adhesion tothe endothelium, the reduction or delay of platelet aggregation, thereduction or delay of the formation of platelet strings, the reductionor delay of thrombus formation, the reduction or delay of thrombusgrowth, the reduction or delay of vessel occlusion, the proteolyticcleavage of vWF, the disintegration of thrombi, or by cleavage of apeptide substrate, for example a FRETS-VWF73 peptide (Kokame et al., BrJ Haematol. 2005 April; 129(1):93-100) or variant thereof.

Likewise, ADAMTS13 proteins may be further modified, for example, bypost-translational modifications (e.g., glycosylation at one or moreamino acids selected from human residues 142, 146, 552, 579, 614, 667,707, 828, 1235, 1354, or any other natural or engineered modificationsite) or by ex vivo chemical or enzymatic modification, includingwithout limitation, glycosylation, modification by water soluble polymer(e.g., PEGylation, sialylation, HESylation, etc.), tagging, and thelike.

As used herein, “one unit of ADAMTS13 activity” is defined as the amountof activity in 1 ml of pooled normal human plasma, regardless of theassay being used. For example, one unit of ADAMTS13 FRETS-VWF73 activityis the amount of activity needed to cleave the same amount ofFRETS-VWF73 substrate (Kokame et al., Br J Haematol. 2005 April;129(1):93-100) as is cleaved by one ml of pooled normal human plasma.

As used herein, the terms “ADAMTS13” and “biologically activederivative”, respectively, also include polypeptides obtained viarecombinant DNA technology. The recombinant ADAMTS13 (“rADAMTS13”), e.g.recombinant human ADAMTS13 (“r-hu-ADAMTS13”), may be produced by anymethod known in the art. One specific example is disclosed in WO02/42441 which is incorporated herein by reference with respect to themethod of producing recombinant ADAMTS13. This may include any methodknown in the art for (i) the production of recombinant DNA by geneticengineering, e.g. via reverse transcription of RNA and/or amplificationof DNA, (ii) introducing recombinant DNA into prokaryotic or eukaryoticcells by transfection, i.e. via electroporation or microinjection, (iii)cultivating said transformed cells, e.g. in a continuous or batchwisemanner, (iv) expressing ADAMTS13, e.g. constitutively or upon induction,and (v) isolating said ADAMTS13, e.g. from the culture medium or byharvesting the transformed cells, in order to (vi) obtain substantiallypurified recombinant ADAMTS13, e.g. via anion exchange chromatography oraffinity chromatography. The term “biologically active derivative”includes also chimeric molecules such as, e.g. ADAMTS13 (or abiologically active derivative thereof) in combination with Ig, in orderto improve the biological/pharmacological properties such as, e.g. halflife of ADAMTS13 in the circulation system of a mammal, particularlyhuman. The Ig could have also the site of binding to an optionallymutated Fc receptor.

As used herein, the term “thrombus” refers to a blood clot, especially aplatelet-comprising blood clot, a microthrombus, and/or an embolus. Saidthrombus may be attached to an arterial or venous blood vessel or not,and may partially or completely block the blood flow in an arterial orvenous blood vessel.

As used herein, the terms “vitamin B3”, “nicotinamide”, “niacinamide”,“niacin”, and “nicotinic acid” may be used interchangeably to refer toany member of the B3 family of vitamins.

As used herein, a “therapeutically effective amount or dose” or“sufficient amount or dose” refers to a dose that produces effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins).

As used herein, a “physiological concentration” of salt refers to a saltconcentration of between about 100 mM and about 200 mM of apharmaceutically acceptable salt. Non-limiting examples ofpharmaceutically acceptable salts include, without limitation, sodiumand potassium chloride, sodium and potassium acetate, sodium andpotassium citrate, sodium and potassium phosphate.

As used herein, a “sub-physiological concentration” of salt refers to asalt concentration of less than about 100 mM of a pharmaceuticallyacceptable salt. In preferred embodiments, a sub-physiologicalconcentration of salt is less than about 80 mM of a pharmaceutical salt.In another preferred embodiment, a sub-physiological concentration ofsalt is less than about 60 mM of a pharmaceutical salt.

As used herein, the term “about” denotes an approximate range of plus orminus 10% from a specified value. For instance, the language “about 20%”encompasses a range of 18-22%. As used herein, about also includes theexact amount. Hence “about 20%” means “about 20%” and also “20%.”

As used herein, “storage” means that a formulation is not immediatelyadministered to a subject once prepared, but is kept for a period oftime under particular conditions (e.g., particular temperature, etc.)prior to use. For example, a liquid or lyophilized formulation can bekept for days, weeks, months or years, prior to administration to asubject under varied temperatures such as refrigerated (0° to 10° C.) orroom temperature (e.g., temperature up to 32° C.).

As used herein, the term “chemically defined medium” refers to asynthetic growth medium in which the identity and concentration of allthe components are known. Chemically defined mediums do not containbacterial, yeast, animal, or plant extracts, although they may or maynot include individual plant or animal-derived components (e.g.,proteins, polypeptides, etc.). Non-limiting examples of commerciallyavailable chemically defined mediums include, various EX-CELL® mediums(SAFC Biosciences, Inc), various Dulbecco's Modified Eagle's (DME)mediums (Sigma-Aldrich Co; SAFC Biosciences, Inc), Ham's NutrientMixture (Sigma-Aldrich Co; SAFC Biosciences, Inc), and the like. Methodsof preparing chemically defined culture mediums are known in the art,for example in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217,and U.S. Patent Application Publication Numbers 2008/0009040 and2007/0212770, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

As used herein, the term “oligopeptide-free culture medium” refers to aprotein-free medium that does not comprise oligopeptides, such as, e.g.,oligopeptides derived from a protein hydrolysate. In one embodiment, themedium does not comprise oligopeptides having twenty or more aminoacids. In one embodiment of the present invention, the medium does notcomprise oligopeptides having fifteen or more amino acids. In anotherembodiment of the invention, the medium does not comprise oligopeptideshaving ten or more amino acids. In one embodiment the medium does notcomprise oligopeptides having seven or more amino acids. In anotherembodiment the medium does not comprise oligopeptides having five ormore amino acids. In still another embodiment the medium does notcomprise oligopeptides having three or more amino acids. According to afurther embodiment of the present invention, the medium does notcomprise oligopeptides having two or more amino acids. Methods ofpreparing oligopeptide-free culture medium are known in the art, forexample in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, andU.S. Patent Application Publication Numbers 2008/0009040 and2007/0212770, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

As used herein, the term “serum-free culture medium” refers to a culturemedium that is not supplemented with an animal serum. Althoughoftentimes serum-free mediums are chemically defined mediums, serum-freemediums may be supplemented with discrete animal or plant proteins orprotein fractions. Methods of preparing serum-free culture medium areknown in the art, for example in U.S. Pat. Nos. 6,171,825 and 6,936,441,WO 2007/077217, and U.S. Patent Application Publication Numbers2008/0009040 and 2007/0212770, the disclosures of which are incorporatedherein by reference in their entireties for all purposes.

As used herein, the term “animal protein-free culture medium” refers toa culture medium that is not supplemented with an animal serum, protein,or protein fraction. Although oftentimes animal protein-free culturemediums are chemically defined mediums, animal protein-free culturemediums may contain plant or yeast hydrolysates. Methods of preparinganimal protein-free culture medium are known in the art, for example inU.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, and U.S. PatentApplication Publication Numbers 2008/0009040 and 2007/0212770, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

III. ADAMTS13 Compositions and Formulation

In one aspect, the present invention provides stabilized formulations ofADAMTS13 (A13) and rADAMTS13 (rA13) proteins. In one embodiment, theformulations of the invention are stable when stored at temperatures upto at least about 40° C. for at least about 6 months. In otherembodiments, the formulations provided herein retain significantADAMTS13 activity when stored for extended periods of time. In yet otherembodiments, the formulations of the invention reduce or retarddimerization, oligomerization, and/or aggregation of an ADAMTS13protein.

In one embodiment, the present invention provides formulations ofADAMTS13 comprising a therapeutically effective amount or dose of anADAMTS13 protein, a sub-physiological to physiological concentration ofa pharmaceutically acceptable salt, a stabilizing concentration of oneor more sugars and/or sugar alcohols, a non-ionic surfactant, abuffering agent providing a neutral pH to the formulation, andoptionally a calcium and/or zinc salt. Generally, the stabilized A13formulations provided herein are suitable for pharmaceuticaladministration. In a preferred embodiment, the A13 protein is humanADAMTS13 or a biologically active derivative or fragment thereof.

In certain embodiments, the ADAMTS13 formulations are liquidformulations. In other embodiments, the ADAMTS13 formulations arelyophilized formulations that are lyophilized from a liquid formulationas provided herein.

In certain embodiments of the formulations provided herein, the ADAMTS13protein is a human ADAMTS13 (hA13) or recombinant human ADAMTS13(rhA13), or a biologically active derivative or fragment thereof. In oneembodiment, the amino acid sequence of hA13 is that of GenBank accessionnumber NP_620594. In another embodiment, the amino acid sequence of hA13comprises amino acids 75 to 1427 of NP_620594, a natural or conservativevariant thereof, or a biologically active fragment thereof.

In certain embodiments, ADAMTS13 is provided in a therapeuticallyeffective dose between about 0.05 mg/mL and about 10 mg/mL. In otherembodiments, ADAMTS13 is present at a concentration of between about 0.1mg/mL and about 10 mg/mL. In yet other embodiments, ADAMTS13 is presentat a concentration of between about 0.1 mg/mL and about 5 mg/mL. Inanother embodiment, ADAMTS13 is present at a concentration of betweenabout 0.1 mg/mL and about 2 mg/mL. In yet other embodiments, ADAMTS13may be present at about 0.01 mg/mL, or at about 0.02 mg/mL, 0.03 mg/mL,0.04 mg/mL, 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL,0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL,1.4 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL, 2.0mg/mL, 2.5 mg/mL, 3.0 mg/mL, 3.5 mg/mL, 4.0 mg/mL, 4.5 mg/mL, 5.0 mg/mL,5.5 mg/mL, 6.0 mg/mL, 6.5 mg/mL, 7.0 mg/mL, 7.5 mg/mL, 8.0 mg/mL, 8.5mg/mL, 9.0 mg/mL, 9.5 mg/mL, 10.0 mg/mL, or a higher concentration. Inone embodiment, the concentration of a relatively pure ADAMTS13formulation may be determined by spectroscopy (i.e., total proteinmeasured at A₂₈₀) or other bulk determination (e.g., Bradford assay,silver stain, weight of a lyophilized powder, etc.). In otherembodiments, the concentration of ADAMTS13 may be determined by anADAMTS13 ELISA assay (e.g., mg/mL antigen).

In yet other embodiments, the concentration of ADAMTS13 in a formulationprovided by the present invention may be expressed as a level ofenzymatic activity. For example, in one embodiment an ADAMTS13formulation may contain between about 10 units of FRETS-VWF73 activityand about 10,000 units of FRETS-VWF73 activity or other suitable A13enzymatic unit (IU). In other embodiments, the formulation may containbetween about 20 units of FRETS-VWF73 (U_(FV73)) activity and about8,000 units of FRETS-VWF73 activity, or between about 30 U_(FV73) andabout 6,000 U_(FV73), or between about 40 U_(FV73) and about 4,000U_(FV73), or between about 50 U_(FV73) and about 3,000 U_(FV73), orbetween about 75 U_(FV73) and about 2,500 U_(FV73), or between about 100U_(FV73) and about 2,000 U_(FV73), or between about 200 U_(FV73) andabout 1,500 U_(FV73), or between about other ranges therein. In apreferred embodiment, an ADAMTS13 formulation provided herein containsbetween about 150 and about 600 U_(FV73). In certain embodiments, aformulation contains about 10 units of FRETS-VWF73 activity, or about20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500,1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500,2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500,3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500,4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400, 5,500,5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500,6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500,7,600, 7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500,8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500,9,600, 9,700, 9,800, 9,900, 10,000 or more units of FRETS-VWF73activity.

Similarly, in certain embodiments, the concentration of ADAMTS13 may beexpressed as an enzymatic activity per unit volume, for example, A13enzymatic units per mL (IU/mL). For example, in one embodiment anADAMTS13 formulation may contain between about 10 IU/mL and about 10,000IU/mL. In other embodiments, the formulation may contain between about20 IU/mL and about 8,000 IU/mL, or between about 30 IU/mL and about6,000 IU/mL, or between about 40 IU/mL and about 4,000 IU/mL, or betweenabout 50 IU/mL and about 3,000 IU/mL, or between about 75 IU/mL andabout 2,500 IU/mL, or between about 100 IU/mL and about 2,000 IU/mL, orbetween about 200 IU/mL and about 1,500 IU/mL, or between about otherranges therein. In a preferred embodiment, an ADAMTS13 formulationprovided herein contains between about 150 IU/mL and about 600 IU/mL. Inanother preferred embodiment, an ADAMTS13 formulation provided hereincontains between about 100 IU/mL and about 1,000 IU/mL. In certainembodiments, a formulation contains about 10 IU/mL, or about 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600,700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700,1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700,2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700,3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700,4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700,5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700,6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600, 7,700,7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600, 8,700,8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700,9,800, 9,900, 10,000 or more IU/mL.

In certain embodiments, the stabilized ADAMTS13 formulations provided bythe present invention will contain a sub-physiological to physiologicalsalt concentration, for example, between and 0 mM and about 200 mM of apharmaceutically acceptable salt. In one embodiment, an ADAMTS13formulation will contain a physiological concentration of salt, forexample, between about 100 mM and about 200 mM of a pharmaceuticallyacceptable salt. In other embodiments, an ADAMTS13 formulation willcontain about 0 mM, or about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160mM, 170 mM, 180 mM, 190 mM, 200 mM, or more of a pharmaceuticallyacceptable salt. In a preferred embodiment, the salt is sodium orpotassium chloride.

Advantageously, it has been found that ADAMTS13 formulations containinga sub-physiological concentration of a pharmaceutically acceptable saltform compact lyocakes with smooth surfaces. Furthermore, it has beenfound that low salt lyophilized formulations of ADAMTS13 proteins reduceprotein aggregation as compared to formulations prepared withphysiological concentrations of salt. Accordingly, in a preferredembodiment, the present invention provides low salt formulations ofADAMTS13 containing a sub-physiological concentration of apharmaceutically acceptable salt, for example, less than about 100 mM ofa pharmaceutically acceptable salt. In one embodiment, a low saltADAMTS13 formulation provided herein contains less than about 100 mM ofa pharmaceutical salt. In a preferred embodiment, a low salt ADAMTS13formulation provided herein contains less than about 80 mM of apharmaceutical salt. In another preferred embodiment, a low saltADAMTS13 formulation provided herein contains less than about 60 mM of apharmaceutical salt (i.e., between about 0 mM and about 60 mM salt). Inanother preferred embodiment, a low salt ADAMTS13 formulation willcontain between about 30 mM and about 60 mM of a pharmaceuticallyacceptable salt. In yet other embodiments, a low salt ADAMTS13formulation will contain about 0 mM, or about 5 mM, 10 mM, 15 mM, 20 mM,25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM of a pharmaceuticallyacceptable salt. In a preferred embodiment, a low salt ADAMTS13formulation is a lyophilized formulation. In a preferred embodiment, thesalt is sodium or potassium chloride.

It has also been found that the inclusion of moderate levels (i.e.,between about 2% and about 6%) of one or more sugars and/or sugaralcohols assists in the preparation of compact lyocakes with smoothsurfaces and helps to stabilize ADAMTS13 upon lyophilization.Accordingly, in one embodiment, the present invention provides ADAMTS13formulations containing between about 2% and about 6% of one or moresugars and/or sugar alcohols. Any sugar such as mono-, di-, orpolysaccharides, or water-soluble glucans, including for examplefructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose,dextran, trehalose, pullulan, dextrin, cyclodextrin, soluble starch,hydroxyethyl starch, and carboxymethylcellulose may be used. In aparticular embodiment, sucrose or trehalose is used as a sugar additive.Sugar alcohols are defined as a hydrocarbon having between about 4 andabout 8 carbon atoms and a hydroxyl group. Non-limiting examples ofsugar alcohols that may be used in the ADAMTS13 formulations providedherein include, mannitol, sorbitol, inositol, galactitol, dulcitol,xylitol, and arabitol. In one embodiment, mannitol is used as a sugaralcohol additive. In a preferred embodiment, an ADAMTS13 formulationcontains both a sugar and a sugar alcohol additive.

The sugars and sugar alcohols may be used individually or incombination. In some embodiments, the sugar, sugar alcohol, orcombination thereof will be present in the formulation at aconcentration of between about 0.5% and about 7%. In one embodiment, thesugar and/or sugar alcohol content of the formulation will be betweenabout 0.5% and about 5%. In certain embodiments, the sugar, sugaralcohol, or combination thereof will be present at a concentration ofbetween about 1% and about 5%. In a preferred embodiment, the sugar,sugar alcohol, or combination thereof will be present at a concentrationof between about 2% and about 6%. In another preferred embodiment, thesugar, sugar alcohol, or combination thereof will be present at aconcentration of between about 3% and about 5%. In certain embodiments,the final concentration may be about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%,4%, 4.5%, 5%, 5.5%, 6.0%, 6.5%, or 7.0% sugar, sugar alcohol, orcombination thereof. In particular embodiments, a formulation providedherein may comprise a sugar at a concentration from about 0.5% to about5.0% and a sugar alcohol at a concentration from about 0.5% to about5.0%. Any combination of sugar and sugar alcohol concentrations may beused, e.g. a sugar present at a concentration of about 0.5%, 1%, 1.5%,2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6.0%, 6.5%, or 7.0% and a sugaralcohol present at a concentration of about 0.5%, 1%, 1.5%, 2%, 2.5%,3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6.0%, 6.5%, or 7.0%.

Advantageously, it was also found that the inclusion of a non-ionicsurfactant substantially reduces the aggregation of ADAMTS13formulations. Accordingly, in one embodiment, ADAMTS13 formulationscontaining a stabilizing concentration of a non-ionic detergent areprovided. Pharmaceutically acceptable nonionic surfactants that may beused in the formulations of the present invention are known in the artof pharmaceutical science, and include, without limitation, Polysorbate80 (Tween 80), Polysorbate 20 (Tween 20), and various poloxamers orpluronics, including Pluronic F-68, and BRIJ 35, or mixtures thereof. Ina preferred embodiment, the nonionic surfactant used in the presentpharmaceutical formulations is Polysorbate 80. In certain embodiments, asurfactant may be used in a formulation provided herein at aconcentration between about 0.001% and about 0.2%. In a preferredembodiment, the surfactant is used at a concentration of between about0.01% and about 0.1%. In another preferred embodiment, the surfactant isused at a concentration of about 0.05%. For example, in certainembodiments, the formulation may include a nonionic surfactant at aconcentration of about 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%,0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.125%, 0.15%, 0.175%, 0.2%,and the like.

Furthermore, it was found that ADAMTS13 formulations were stabilizedwhen formulated at a neutral pH between about 6.5 and about 7.5.Accordingly, in certain embodiments, ADAMTS13 formulations are providedthat contain a buffering agent suitable to maintain the formulation at aneutral pH. Pharmaceutically acceptable buffering agents are well knownin the art, and include without limitation, phosphate buffers,histidine, sodium citrate, HEPES, Tris, Bicine, glycine,N-glycylglycine, sodium acetate, sodium carbonate, glycylglycine,lysine, arginine, sodium phosphate, and mixtures thereof. In preferredembodiments, the buffer is selected from histidine, phosphate buffer,HEPES, and sodium citrate. In one preferred embodiment, the buffer ishistidine or HEPES. In a specific embodiment, the buffer in histidine.In another specific embodiment, the buffer is HEPES. In one embodiment,the pH of the formulations provided herein is between about 6.5 andabout 9.0. In certain embodiments, the pH of the formulation is about6.5 or about 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In apreferred embodiment, the pH of the A13 formulation is between about 6.0and about 8.0. In a more preferred embodiment, the pH of the A13formulation is between about 6.5 and about 7.5. In a particularembodiment, the pH of the A13 formulation is about 7.0. In anotherparticular embodiment, the pH of the A13 formulation is 7.0±0.2.

It is also demonstrated herein that the inclusion of calcium furtherstabilizes formulations of ADAMTS13. Accordingly, in certainembodiments, stabilized ADAMTS13 formulations are provided which containbetween about 0.5 mM and about 20 mM calcium (e.g., calcium chloride).Any pharmaceutically acceptable calcium salt may be used in theformulations provided herein. Non-limiting examples of calcium salt thatmay be used include, for example, CaCl₂, CaCO₃, Ca(C₆H₁₁O₇)₂, Ca₃(PO₄)₂,Ca(C₁₈H₃₅O₂)₂, and the like. In one embodiment, calcium is present in anADAMTS13 formulation of the invention at a concentration from about 0.5mM to about 10 mM. In another embodiment, calcium is present in anADAMTS13 formulation at a concentration between about 2 mM and about 5mM. In a preferred embodiment, calcium is present in an ADAMTS13formulation at a concentration from about 2 mM to about 4 mM. In certainembodiments, the concentration of calcium is about 0.5 mM, or about 1mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM,13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM. In aparticular embodiment, the concentration of calcium is about 2 mM. Inanother preferred embodiment, the concentration of calcium is about 3mM. In yet another preferred embodiment, the concentration of calcium isabout 4 mM.

Similarly, it has been found that under certain conditions, theinclusion of zinc further stabilizes an ADAMTS13 formulation as providedherein. For example, FIG. 34 shows that inclusion of between about 2 μMand about 10 μM zinc further stabilized calcium containing ADAMTS13formulations. Any pharmaceutically acceptable zinc salt may be used inthe formulations provided herein. Non-limiting examples of zinc saltthat may be used include, for example, ZnSO₄.7H₂O, ZnSO₃.2H₂O,Zn₃(PO₄)₂, and (C₆H₅O₇)₂Zn₃.2H₂O, and the like. In one embodiment, ZnSO₄is used in the ADAMTS13 formulations provided herein. In someembodiments, zinc is present in an ADAMTS13 formulation of the inventionat a concentration from about 0.5 μM to about 20.0 In a preferredembodiment, zinc is included in an ADAMTS13 formulation at aconcentration of between about 0.5 μM to about 10.0 μM. In certainembodiments, the concentration of zinc is about 0.5 μM, or about 1 μM, 2μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM.

In some embodiments, the ADAMTS13 formulations provided herein mayfurther comprise one or more pharmaceutically acceptable excipients,carriers, and/or diluents. In addition, the formulations provided hereinmay further comprise other medicinal agents, carriers, adjuvants,diluents, tissue permeation enhancers, solubilizers, and the like.Methods for preparing compositions and formulations for pharmaceuticaladministration are known to those skilled in the art (see, for example,R EMINGTON'S P HARMACEUTICAL S CIENCES, 18TH ED., Mack Publishing Co.,Easton, Pa. (1990)).

In one embodiment, the ADAMTS13 formulations provided herein will have atonocity in a range between about 200 mOsmol/L and about 400 mOsmol/L,or in a range between about 250 and about 350 mOsmol/L. In certainembodiments, an ADAMTS13 formulation provided herein will have atonocity, for example, of about 200 mOsmol/L, or of about 210 mOsmol/L,220 mOsmol/L, 230 mOsmol/L, 240 mOsmol/L, 250 mOsmol/L, 260 mOsmol/L,270 mOsmol/L, 280 mOsmol/L, 290 mOsmol/L, 300 mOsmol/L, 310 mOsmol/L,320 mOsmol/L, 330 mOsmol/L, 340 mOsmol/L, 350 mOsmol/L, 360 mOsmol/L,370 mOsmol/L, 380 mOsmol/L, 390 mOsmol/L, or 400 mOsmol/L.

Examples of tonocity agents that may be used in the formulationsprovided herein include, without limitation, sodium chloride, dextrose,sucrose, xylitol, fructose, glycerol, sorbitol, mannitol, trehalose,potassium chloride, mannose, calcium chloride, magnesium chloride, otherinorganic salts, other sugars, other sugar alcohols, and combinationsthereof. In certain embodiments, an ADAMTS13 formulation may comprise atleast one tonocity agent, or at least two, three, four, five, or moretonocity agents.

The ADAMTS13 formulations provided herein may be formulated foradministration via known methods, such as intravenous administration,e.g., as a bolus or by continuous infusion over a period of time, byintramuscular, intraperitoneal, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes. In certain embodiments, the ADAMTS13 formulationsprovided herein can be administered either systemically or locally.Systemic administration includes, without limitation: oral, subdermal,intraperitioneal, subcutaneous, transnasal, sublingual, or rectal routesof administration. Local administration includes, without limitation:topical, subcutaneous, intramuscular, and intraperitoneal routes ofadministration.

In one aspect of the invention, a composition of monomeric ADAMTS13protein is provided. In certain embodiments, the composition ofmonomeric ADAMTS13 protein is substantially free of aggregated ADAMTS13,dimeric ADAMTS13, or both aggregated and dimeric ADAMTS13. In someembodiments, the monomeric composition has a higher specific activitythan a similar composition containing aggregated and/or dimeric ADAMTS13protein. In a particular embodiment, the monomeric ADAMTS13 compositionis produced by a method comprising gel filtration or size exclusionchromatography. In one particular embodiment, the ADAMTS13 protein is ahuman ADAMTS13 (hA13) or recombinant human ADAMTS13 (rhA13), or abiologically active derivative or fragment thereof.

In another aspect, formulations of monomeric ADAMTS13 compositions areprovided. In one embodiment, the formulation is a pharmaceuticallyacceptable formulation of the monomeric ADAMTS13 protein. In certainembodiments, the formulation is substantially free of aggregatedADAMTS13, dimeric ADAMTS13, or both aggregated and dimeric ADAMTS13. Insome embodiments, the monomeric ADAMTS13 formulations have a higherspecific activity than similar formulations containing aggregated and/ordimeric ADAMTS13 protein. In a particular embodiment, the monomericADAMTS13 formulation is produced by a method comprising gel filtrationor size exclusion chromatography. In one particular embodiment, theADAMTS13 protein in the formulation is a human ADAMTS13 (hA13) orrecombinant human ADAMTS13 (rhA13), or a biologically active derivativeor fragment thereof. In certain embodiments, the monomeric ADAMTS13protein is formulated according to a formulation provided herein.

In embodiment, the present invention provides formulations of ADAMTS13comprising from about 0.0.5 mg/ml to about 10.0 mg/ml ADAMTS13 protein,from about 0 mM to about 200 mM of a pharmaceutically acceptable salt, asugar and/or sugar alcohol, a non-ionic surfactant, and a bufferingagent. In certain embodiments, the formulations may further comprisecalcium and/or zinc. In other embodiments, the formulation may bebuffered at a pH of between about 6.5 and 9.0. In certain embodiments,the A13 formulations are suitable for pharmaceutical administration.

In a specific embodiment, the present invention provides a stabilizedformulation of ADAMTS13, comprising: 0.05 mg/ml to 10.0 mg/ml ADAMTS13;0 mM to 200 mM of a pharmaceutically acceptable salt; 0.5 mM to 20 mMcalcium; a sugar and/or sugar alcohol; a nonionic surfactant; and abuffering agent for maintaining a pH of between about 6.5 and about 7.5.In a preferred embodiment, the pharmaceutically acceptable salt issodium chloride or potassium chloride.

In one embodiment, a stabilized ADAMTS13 formulation is providedcomprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13; 0 mM to 200 mM of apharmaceutically acceptable salt; 0.5 mM to 20 mM calcium; a sugarand/or sugar alcohol; a nonionic surfactant; and a buffering agent formaintaining a pH of between about 6.5 and about 7.5 contains betweenabout 50 units per mL and about 1000 units per mL of ADAMTS13 activity.In a preferred embodiment, the pharmaceutically acceptable salt issodium chloride or potassium chloride.

In another embodiment, the present invention provides stabilizedADAMTS13 formulations comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13; 0 mMto 200 mM of a pharmaceutically acceptable salt; 1 mM to 10 mM calcium;a sugar and/or sugar alcohol; a nonionic surfactant; and a bufferingagent for maintaining a pH of between about 6.5 and about 7.5. In apreferred embodiment, the formulation contains between about 2 mM andabout 4 mM calcium. In a preferred embodiment, the pharmaceuticallyacceptable salt is sodium chloride or potassium chloride.

In another embodiment, the present invention provides stabilizedADAMTS13 formulations comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13; 0 mMto 200 mM of a pharmaceutically acceptable salt; 0.5 mM to 20 mMcalcium; between about 2% and about 6% of a sugar and/or sugar alcohol;a nonionic surfactant; and a buffering agent for maintaining a pH ofbetween about 6.5 and about 7.5. In certain embodiments, the sugarand/or sugar alcohol is selected from the group consisting of sucrose,trehalose, mannitol, and a combination thereof. In a preferredembodiment, the sugar and/or sugar alcohol is a mixture of sucrose andmannitol. In a particular embodiment, the mixture of sucrose andmannitol consists of about 1% sucrose and about 3% mannitol. In certainembodiments, the formulation comprises between about 1 mM and about 10mM calcium, preferably between about 2 mM and about 4 mM calcium. In apreferred embodiment, the pharmaceutically acceptable salt is sodiumchloride or potassium chloride.

In another embodiment, the present invention provides stabilizedADAMTS13 formulations comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13; 0 mMto 200 mM of a pharmaceutically acceptable salt; 0.5 mM to 20 mMcalcium; a sugar and/or sugar alcohol; between about 0.01% and 0.1% of anonionic surfactant; and a buffering agent for maintaining a pH ofbetween about 6.5 and about 7.5. In certain embodiments, the surfactantis selected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, BRIJ 35, and a combination thereof. In a preferredembodiment, the sugar and/or sugar alcohol is a mixture of sucrose andmannitol. In a particular embodiment, the surfactant is Polysorbate 80.In certain embodiments, the formulation comprises between about 1 mM andabout 10 mM calcium, preferably between about 2 mM and about 4 mMcalcium. In a preferred embodiment, the pharmaceutically acceptable saltis sodium chloride or potassium chloride.

In another embodiment, the present invention provides stabilizedADAMTS13 formulations comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13; 0 mMto 200 mM of a pharmaceutically acceptable salt; 0.5 mM to 20 mMcalcium; a sugar and/or sugar alcohol; a nonionic surfactant; and abuffering agent for maintaining a pH of between about 6.5 and about 7.5,wherein the buffering agent is histidine or HEPES. In certainembodiments, the buffering agent is present at a concentration betweenabout 5 mM and about 100 mM, preferably between about 10 mM and about 50mM. In another preferred embodiment, the pH of the formulation is7.0±0.2. In certain embodiments, the formulation comprises between about1 mM and about 10 mM calcium, preferably between about 2 mM and about 4mM calcium. In a preferred embodiment, the pharmaceutically acceptablesalt is sodium chloride or potassium chloride.

In another embodiment, the present invention provides stabilizedADAMTS13 formulations comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13; 0 mMto 200 mM of a pharmaceutically acceptable salt; 0.5 mM to 20 mMcalcium; a sugar and/or sugar alcohol; a nonionic surfactant; and abuffering agent for maintaining a pH of between about 6.5 and about 7.5.In certain embodiments, the formulation further comprises between about0.5 μM and about 20 μM zinc. In certain embodiments, the formulationcomprises between about 1 mM and about 10 mM calcium, preferably betweenabout 2 mM and about 4 mM calcium. In a preferred embodiment, thepharmaceutically acceptable salt is sodium chloride or potassiumchloride.

In a preferred embodiment, the present invention provides a stabilizedADAMTS13 formulation comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13; 0 to60 mM NaCl; 2 mM to 4 mM calcium; 2% to 4% mannitol; 0.5% to 2% sucrose;0.025 to 0.1% Polysorbate 80; and 10 mM to 50 mM histidine (pH 7.0±0.2).In one embodiment, the formulation further comprises between about 0.5μM and about 20 μM zinc.

In another embodiment, stabilized low salt ADAMTS13 formulations areprovided comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13; less than about100 mM of a pharmaceutically acceptable salt; 0.5 mM to 20 mM calcium; asugar and/or sugar alcohol; a nonionic surfactant; and a buffering agentfor maintaining a pH of approximately between 6.5 and 7.5. In apreferred embodiment, the pharmaceutically acceptable salt is sodiumchloride or potassium chloride. In a preferred embodiment, the low saltADAMTS13 formulation is a lyophilized formulation.

In one embodiment, a stabilized low salt ADAMTS13 formulation isprovided comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13; less than about100 mM of a pharmaceutically acceptable salt; 0.5 mM to 20 mM calcium; asugar and/or sugar alcohol; a nonionic surfactant; and a buffering agentfor maintaining a pH of between about 6.5 and about 7.5 contains betweenabout 50 units per mL and about 1000 units per mL of ADAMTS13 activity.In a preferred embodiment, the pharmaceutically acceptable salt issodium chloride or potassium chloride. In a preferred embodiment, thelow salt ADAMTS13 formulation is a lyophilized formulation.

In another embodiment, the present invention provides stabilized lowsalt ADAMTS13 formulations comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13;less than about 100 mM of a pharmaceutically acceptable salt; 1 mM to 10mM calcium; a sugar and/or sugar alcohol; a nonionic surfactant; and abuffering agent for maintaining a pH of between about 6.5 and about 7.5.In a preferred embodiment, the formulation contains between about 2 mMand about 4 mM calcium. In a preferred embodiment, the pharmaceuticallyacceptable salt is sodium chloride or potassium chloride. In a preferredembodiment, the low salt ADAMTS13 formulation is a lyophilizedformulation.

In another embodiment, the present invention provides stabilized lowsalt ADAMTS13 formulations comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13;less than about 100 mM of a pharmaceutically acceptable salt; 0.5 mM to20 mM calcium; between about 2% and about 6% of a sugar and/or sugaralcohol; a nonionic surfactant; and a buffering agent for maintaining apH of between about 6.5 and about 7.5. In certain embodiments, the sugarand/or sugar alcohol is selected from the group consisting of sucrose,trehalose, mannitol, and a combination thereof. In a preferredembodiment, the sugar and/or sugar alcohol is a mixture of sucrose andmannitol. In a particular embodiment, the mixture of sucrose andmannitol consists of about 1% sucrose and about 3% mannitol. In certainembodiments, the formulation comprises between about 1 mM and about 10mM calcium, preferably between about 2 mM and about 4 mM calcium. In apreferred embodiment, the pharmaceutically acceptable salt is sodiumchloride or potassium chloride. In a preferred embodiment, the low saltADAMTS13 formulation is a lyophilized formulation.

In another embodiment, the present invention provides stabilized lowsalt ADAMTS13 formulations comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13;less than about 100 mM of a pharmaceutically acceptable salt; 0.5 mM to20 mM calcium; a sugar and/or sugar alcohol; between about 0.01% and0.1% of a nonionic surfactant; and a buffering agent for maintaining apH of between about 6.5 and about 7.5. In certain embodiments, thesurfactant is selected from the group consisting of Polysorbate 20,Polysorbate 80, Pluronic F-68, BRIJ 35, and a combination thereof. In apreferred embodiment, the sugar and/or sugar alcohol is a mixture ofsucrose and mannitol. In a particular embodiment, the surfactant isPolysorbate 80. In certain embodiments, the formulation comprisesbetween about 1 mM and about 10 mM calcium, preferably between about 2mM and about 4 mM calcium. In a preferred embodiment, thepharmaceutically acceptable salt is sodium chloride or potassiumchloride. In a preferred embodiment, the low salt ADAMTS13 formulationis a lyophilized formulation.

In another embodiment, the present invention provides stabilized lowsalt ADAMTS13 formulations comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13;less than about 100 mM of a pharmaceutically acceptable salt; 0.5 mM to20 mM calcium; a sugar and/or sugar alcohol; a nonionic surfactant; anda buffering agent for maintaining a pH of between about 6.5 and about7.5, wherein the buffering agent is histidine or HEPES. In certainembodiments, the buffering agent is present at a concentration betweenabout 5 mM and about 100 mM, preferably between about 10 mM and about 50mM. In another preferred embodiment, the pH of the formulation is7.0±0.2. In certain embodiments, the formulation comprises between about1 mM and about 10 mM calcium, preferably between about 2 mM and about 4mM calcium. In a preferred embodiment, the pharmaceutically acceptablesalt is sodium chloride or potassium chloride. In a preferredembodiment, the low salt ADAMTS13 formulation is a lyophilizedformulation.

In another embodiment, the present invention provides stabilized lowsalt ADAMTS13 formulations comprising 0.05 mg/ml to 10.0 mg/ml ADAMTS13;less than about 100 mM of a pharmaceutically acceptable salt; 0.5 mM to20 mM calcium; a sugar and/or sugar alcohol; a nonionic surfactant; anda buffering agent for maintaining a pH of between about 6.5 and about7.5. In certain embodiments, the formulation further comprises betweenabout 0.5 μM and about 20 μM zinc. In certain embodiments, theformulation comprises between about 1 mM and about 10 mM calcium,preferably between about 2 mM and about 4 mM calcium. In a preferredembodiment, the pharmaceutically acceptable salt is sodium chloride orpotassium chloride. In a preferred embodiment, the low salt ADAMTS13formulation is a lyophilized formulation.

In a preferred embodiment, the present invention provides a stabilizedlow salt ADAMTS13 formulation comprising 0.05 mg/ml to 10.0 mg/mlADAMTS13; less than about 100 mM NaCl; 2 mM to 4 mM calcium; 2% to 4%mannitol; 0.5% to 2% sucrose; 0.025 to 0.1% Polysorbate 80; and 10 mM to50 mM histidine (pH 7.0±0.2). In one embodiment, the formulation furthercomprises between about 0.5 μM and about 20 μM zinc. In a preferredembodiment, the low salt ADAMTS13 formulation is a lyophilizedformulation.

In one embodiment, the present invention provides a stabilizedformulation of ADAMTS13 (A13) comprising (a) at least 100 units ADAMTS13activity (i.e., FRETS-vWF73 activity) per mg ADAMTS13; (b) 0 mM to 200mM of a pharmaceutically acceptable salt; (c) 0.5 mM to 20 mM calcium;(d) a sugar and/or sugar alcohol; (e) a nonionic surfactant; and (f) abuffering agent for maintaining a pH between 6.0 and 8.0. In oneembodiment, the stabilized formulation of ADAMTS13 comprises at least200 units A13 activity per mg ADAMTS13. In another embodiment, thestabilized formulation of ADAMTS13 comprises at least 400 units A13activity per mg ADAMTS13. In a preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 600 units A13 activity per mgADAMTS13. In a more preferred embodiment, the stabilized formulation ofADAMTS13 comprises at least 800 units A13 activity per mg ADAMTS13. Inyet another preferred embodiment, the stabilized formulation of ADAMTS13comprises at least 1000 units A13 activity per mg ADAMTS13. In oneembodiment, the stabilized formulation of ADAMTS13 comprises betweenabout 100 units and about 2000 units of ADAMTS13 activity per mgADAMTS13.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 1.0 mM and about 10.0 mM calcium. In a preferredembodiment, the formulation contains between about 2.0 and about 4.0 mMcalcium.

In another embodiment of the stabilized A13 formulation, the formulationcomprises between about 2% and about 6% of a sugar and/or sugar alcohol.In a preferred embodiment, the formulation comprises between about 3%and about 5% of a sugar and/or sugar alcohol. In a specific embodiment,the formulation comprises about 4% of a sugar and/or sugar alcohol. Inone embodiment, the sugar and/or sugar alcohol is selected from thegroup consisting of sucrose, trehalose, mannitol, and a combinationthereof. In a preferred embodiment, the sugar and/or sugar alcohol is amixture of sucrose and mannitol.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 0.01% and about 0.1% of a non-ionic surfactant.In a preferred embodiment, the formulation comprises about 0.05% of anon-ionic surfactant. In one embodiment, the surfactant is selected fromthe group consisting of Polysorbate 20, Polysorbate 80, Pluronic F-68,and BRIJ 35. In a preferred embodiment, the surfactant is Polysorbate80.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 5 mM and about 100 mM of a buffering agent. In apreferred embodiment, the formulation comprises between about 10 mM andabout 50 mM of a buffering agent. In another embodiment, the bufferingagent is histidine or HEPES. In a preferred embodiment, the bufferingagent is histidine. In one embodiment, the pH of the formulation isbetween about 6.5 and 7.5. In a preferred embodiment, the pH of theformulation is 7.0±0.2.

In one embodiment of the stabilized A13 formulation, the formulationfurther comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedformulation of ADAMTS13 (A13) comprising (a) at least 100 units ADAMTS13activity per mg ADAMTS13; (b) 0 to 200 mM NaCl; (c) 2 mM to 4 mMcalcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a stabilized low saltformulation of ADAMTS13 (A13) comprising (a) at least 100 units ADAMTS13activity (i.e., FRETS-vWF73 activity) per mg ADAMTS13; (b) 0 mM to 100mM of a pharmaceutically acceptable salt; (c) 0.5 mM to 20 mM calcium;(d) a sugar and/or sugar alcohol; (e) a nonionic surfactant; and (f) abuffering agent for maintaining a pH between 6.0 and 8.0. In oneembodiment, the stabilized formulation of ADAMTS13 comprises at least200 units A13 activity per mg ADAMTS13. In another embodiment, thestabilized formulation of ADAMTS13 comprises at least 400 units A13activity per mg ADAMTS13. In a preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 600 units A13 activity per mgADAMTS13. In a more preferred embodiment, the stabilized formulation ofADAMTS13 comprises at least 800 units A13 activity per mg ADAMTS13. Inyet another preferred embodiment, the stabilized formulation of ADAMTS13comprises at least 1000 units A13 activity per mg ADAMTS13. In oneembodiment, the stabilized formulation of ADAMTS13 comprises betweenabout 100 units and about 2000 units of ADAMTS13 activity per mgADAMTS13.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 1.0 mM and about 10.0 mM calcium. Ina preferred embodiment, the formulation contains between about 2.0 andabout 4.0 mM calcium.

In another embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 2% and about 6% of a sugar and/orsugar alcohol. In a preferred embodiment, the formulation comprisesbetween about 3% and about 5% of a sugar and/or sugar alcohol. In aspecific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 0.01% and about 0.1% of a non-ionicsurfactant. In a preferred embodiment, the formulation comprises about0.05% of a non-ionic surfactant. In one embodiment, the surfactant isselected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, and BRIJ 35. In a preferred embodiment, the surfactant isPolysorbate 80.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 5 mM and about 100 mM of a bufferingagent. In a preferred embodiment, the formulation comprises betweenabout 10 mM and about 50 mM of a buffering agent. In another embodiment,the buffering agent is histidine or HEPES. In a preferred embodiment,the buffering agent is histidine. In one embodiment, the pH of theformulation is between about 6.5 and 7.5. In a preferred embodiment, thepH of the formulation is 7.0±0.2.

In one embodiment of the stabilized low salt A13 formulation, theformulation further comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlow salt formulation of ADAMTS13 (A13) comprising (a) at least 100 unitsADAMTS13 activity per mg ADAMTS13; (b) 0 to 60 mM NaCl; (c) 2 mM to 4 mMcalcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In a related embodiment, the present invention provides a stabilizedlyophilized formulation of ADAMTS13 (A13), wherein the formulation islyophilized from a liquid formulation comprising (a) at least 100 unitsADAMTS13 activity (i.e., FRETS-vWF73 activity) per mg ADAMTS13; (b) 0 mMto 200 mM of a pharmaceutically acceptable salt; (c) 0.5 mM to 20 mMcalcium; (d) a sugar and/or sugar alcohol; (e) a nonionic surfactant;and (f) a buffering agent for maintaining a pH between 6.0 and 8.0. Inone embodiment, the stabilized formulation of ADAMTS13 comprises atleast 200 units A13 activity per mg ADAMTS13. In another embodiment, thestabilized formulation of ADAMTS13 comprises at least 400 units A13activity per mg ADAMTS13. In a preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 600 units A13 activity per mgADAMTS13. In a more preferred embodiment, the stabilized formulation ofADAMTS13 comprises at least 800 units A13 activity per mg ADAMTS13. Inyet another preferred embodiment, the stabilized formulation of ADAMTS13comprises at least 1000 units A13 activity per mg ADAMTS13. In oneembodiment, the stabilized formulation of ADAMTS13 comprises betweenabout 100 units and about 2000 units of ADAMTS13 activity per mgADAMTS13.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 1.0 mM and about 10.0 mM calcium. Ina preferred embodiment, the formulation contains between about 2.0 andabout 4.0 mM calcium.

In another embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 2% and about 6% of a sugar and/orsugar alcohol. In a preferred embodiment, the formulation comprisesbetween about 3% and about 5% of a sugar and/or sugar alcohol. In aspecific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 0.01% and about 0.1% of a non-ionicsurfactant. In a preferred embodiment, the formulation comprises about0.05% of a non-ionic surfactant. In one embodiment, the surfactant isselected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, and BRIJ 35. In a preferred embodiment, the surfactant isPolysorbate 80.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 5 mM and about 100 mM of a bufferingagent. In a preferred embodiment, the formulation comprises betweenabout 10 mM and about 50 mM of a buffering agent. In another embodiment,the buffering agent is histidine or HEPES. In a preferred embodiment,the buffering agent is histidine. In one embodiment, the pH of theformulation is between about 6.5 and 7.5. In a preferred embodiment, thepH of the formulation is 7.0±0.2.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation further comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlyophilized formulation of ADAMTS13 (A13), wherein the formulation islyophilized from a liquid formulation comprising (a) at least 100 unitsADAMTS13 activity per mg ADAMTS13; (b) 0 to 200 mM NaCl; (c) 2 mM to 4mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In a related embodiment, the present invention provides a stabilized lowsalt lyophilized formulation of ADAMTS13 (A13), wherein the formulationis lyophilized from a liquid formulation comprising (a) at least 100units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mg ADAMTS13;(b) 0 mM to 100 mM of a pharmaceutically acceptable salt; (c) 0.5 mM to20 mM calcium; (d) a sugar and/or sugar alcohol; (e) a nonionicsurfactant; and (f) a buffering agent for maintaining a pH between 6.0and 8.0. In one embodiment, the stabilized formulation of ADAMTS13comprises at least 200 units A13 activity per mg ADAMTS13. In anotherembodiment, the stabilized formulation of ADAMTS13 comprises at least400 units A13 activity per mg ADAMTS13. In a preferred embodiment, thestabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 1.0 mM and about10.0 mM calcium. In a preferred embodiment, the formulation containsbetween about 2.0 and about 4.0 mM calcium.

In another embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 2% and about 6% ofa sugar and/or sugar alcohol. In a preferred embodiment, the formulationcomprises between about 3% and about 5% of a sugar and/or sugar alcohol.In a specific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 0.01% and about0.1% of a non-ionic surfactant. In a preferred embodiment, theformulation comprises about 0.05% of a non-ionic surfactant. In oneembodiment, the surfactant is selected from the group consisting ofPolysorbate 20, Polysorbate 80, Pluronic F-68, and BRIJ 35. In apreferred embodiment, the surfactant is Polysorbate 80.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 5 mM and about 100mM of a buffering agent. In a preferred embodiment, the formulationcomprises between about 10 mM and about 50 mM of a buffering agent. Inanother embodiment, the buffering agent is histidine or HEPES. In apreferred embodiment, the buffering agent is histidine. In oneembodiment, the pH of the formulation is between about 6.5 and 7.5. In apreferred embodiment, the pH of the formulation is 7.0±0.2.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation further comprises between about 0.5 μM and20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlow salt lyophilized formulation of ADAMTS13 (A13), wherein theformulation is lyophilized from a liquid formulation comprising (a) atleast 100 units ADAMTS13 activity per mg ADAMTS13; (b) 0 to 60 mM NaCl;(c) 2 mM to 4 mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose;(f) 0.025 to 0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH7.0±0.2).

IV. Stability of ADAMTS13 Formulations

In certain embodiments, the ADAMTS13 formulations provided herein may bestable for an extended period of time when stored at a certaintemperature, for example, at about −80° C., −20° C., 4° C., 18° C., roomtemperature, 25° C., 30° C., 35° C., 37° C., 40° C., or higher. In someembodiments, an extended period of time is at least about a week. Inother embodiments, an extended period of time may comprise at leastabout 2 weeks, or at least about 3 week, or at least about 1 month, orat least about 2 months, or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 14, 16, or 18 months. In yet other embodiments, the formulations ofthe invention may be stable for at least about 2, 3, 4, 5 or more years.

In certain embodiments of the invention, stability may be measured byone or more biophysical and/or enzymatic properties of the A13 or rA13protein in the formulation. Non-limiting examples of properties that maybe used to assess stability include, enzymatic activity, specificactivity, the degree of mono- or poly-dispersity of the protein, theextent of dimerization, oligomerization, or aggregation of the protein,the degree of unfolding of the protein.

One skilled in the art will readily know of other measures of stabilitythat may be employed to asses the stability of an ADAMTS13 formulation,including without limitation, gel filtration, dynamic or static lightscattering, analytical ultracentrifugation, rp-HPLC, ion-exchangechromatography, fourier transform infrared spectroscopy, calorimetry,differential scanning calorimetry, NMR spectroscopy, mass spectrometry,small angle x-ray scattering (SAX), polyacrylamide gel electrophoresis,and the like.

In one embodiment, the present invention provides formulations of A13 orrA13 that retain at least about 50% of the total ADAMTS13 activity afterstorage for an extended period of time. In other embodiments, aformulation is provided that retains at least about 60%, or at leastabout 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% of the total ADAMTS13 activity after storage for an extended periodof time.

In another embodiment, formulations are provided that maintain at leastabout 50% of the ADAMTS13 specific activity after storage for anextended period of time. In other embodiments, a formulation is providedthat retains at least about 60%, or at least about 70%, 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ADAMTS13 specificactivity after storage for an extended period of time.

In other embodiments, the formulations of the invention will possess aspecific activity of at least about 600U of FRETS-VWF73 activity per mgADAMTS13 protein after storage for an extended period of time. In otherembodiments, a formulation provided herein will have at least about 700U/mg A13, or at least about 800 U/mg, 900 U/mg, 1000 U/mg, 1100 U/mg,1200 U/mg, 1300 U/mg, 1400 U/mg, 1500 U/mg, or higher specific activityafter storage for an extended period of time.

In one embodiment, an A13 formulation provided herein will have apolydispersity, as determined by dynamic light scattering analysis, ofless than about 50% after storage for an extended period of time. Inother embodiments, an A13 formulation will have a polydispersity of lessthan about 45%, or of less than about 40%, 35%, 30%, 25%, 20%, 15%, 10%,5% polydispersity, as measured by dynamic light scattering analysisafter storage for an extended period of time.

In another embodiment of the invention, an A13 formulation providedherein will have an A13 protein population consisting of at least about50% A13 monomers after storage for an extended period of time. In otherembodiments, a formulation may have at least about 60% A13 monomers, orat least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or ahigher percentage of A13 monomers.

V. ADAMTS13 Expression and Purification

In certain embodiments, an ADAMTS13 protein used in the formulationsprovided herein may be expressed, produced, or purified according to amethod disclosed previously, for example, in U.S. Pat. No. 6,926,894, US2005/0266528, US 2007/0015703, U.S. patent application Ser. No.12/437,384, U.S. patent application Ser. No. 12/847,999, and WO2002/42441, all of which are hereby incorporated by reference in theirentirety for all purposes.

A. Host Cells and Vectors

Recombinant ADAMTS proteins can be produced by expression in anysuitable prokaryotic or eukaryotic host system. Examples of eukaryoticcells include, without limitation, mammalian cells, such as CHO, COS,HEK 293, BHK, SK-Hep, and HepG2; insect cells, for example SF9 cells,SF21 cells, S2 cells, and High Five cells; and yeast cells, for exampleSaccharomyces or Schizosaccharomyces cells. In one embodiment, theADAMTS proteins can be expressed in bacterial cells, yeast cells, insectcells, avian cells, mammalian cells, and the like. For example, in ahuman cell line, a hamster cell line, or a murine cell line. In oneparticular embodiment, the cell line is a CHO, BHK, or HEK cell line. Ina preferred embodiment, the cell line is a CHO cell line.

In one embodiment, the cells may be any mammalian cell that can becultured, preferably in a manufacturing process (i.e., at least 1liter), to produce a desired ADAMTS protein such as ADAMTS13. Examplesinclude the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/−DHFR, such as the DUKX-B11 subclone (CHO, Uriaub andChasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse Sertoli cells(TM4, Mather, Biol. Reprod, 23:243-251 (1980)); monkey kidney cells (CV1ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N. Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells;FS4 cells; and the human hepatoma line (Hep G2). Preferably, the cellline is a rodent cell line, especially a hamster cell line such as CHOor BHK.

A wide variety of vectors can be used for the expression of an ADAMTSprotein (e.g., ADAMTS13) and can be selected from eukaryotic andprokaryotic expression vectors. In certain embodiments, a plasmid vectoris contemplated for use in expressing an ADAMTS protein (e.g.,ADAMTS13). In general, plasmid vectors containing replicon and controlsequences which are derived from species compatible with the host cellare used in connection with these hosts. The vector can carry areplication site, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells. The plasmid willcomprise a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) operable linked to one or more control sequences, for example,a promoter.

A preferred method of preparing stable CHO cell clones expressing arecombinant ADAMTS protein is as follows. A DHFR deficient CHO cell lineDUKX-B11 is transfected with a DHFR expression vector to allow forexpression of the relevant recombinant protein, essentially as describedin U.S. Pat. No. 5,250,421 (Kaufman et al., Genetics Institute, Inc.).Selection is carried out by growth in Hypoxanthine/Thymidine (HT) freemedia and amplification of the relevant region coding for expression ofthe recombinant ADAMTS protein and DHFR gene is achieved by propagationof the cells in increasing concentrations of methotrexate. Whereappropriate, CHO cell lines may be adapted for growth in serum and/orprotein free medium, essentially as described in U.S. Pat. No. 6,100,061(Reiter et al, lmmuno Aktiengesellschaft).

In another preferred embodiment, stable HEK293 cells are prepared bytransfecting with a construct containing a hygromycin selectable markerand selecting transformants by antibiotic resistance.

The ability of certain viruses to infect cells or enter cells viareceptor-mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Accordingly, in certain embodiments, a viral vector isused to introduce a nucleotide sequence encoding an ADAMTS protein(e.g., ADAMTS13) into a host cell for expression. The viral vector willcomprise a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) operable linked to one or more control sequences, for example,a promoter. Alternatively, the viral vector may not contain a controlsequence and will instead rely on a control sequence within the hostcell to drive expression of the ADAMTS protein. Non-limiting examples ofvirus vectors that may be used to deliver a nucleic acid includeAdenoviral vectors, AAV vectors, and Retroviral vectors.

In one embodiment, an Adenovirus expression vector include thoseconstructs containing adenovirus sequences sufficient to supportpackaging of the construct and to ultimately express an ADAMTS constructthat has been cloned therein. Adenoviral vectors allow for theintroduction of foreign sequences up to 7 kb (Grunhaus et al., Seminarin Virology, 200(2):535-546, 1992)).

In another embodiment, an adeno-associated virus (AAV) can be used tointroduce a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) into a host cell for expression. AAV systems have beendescribed previously and are generally well known in the art (Kelleherand Vos, Biotechniques, 17(6):1110-7, 1994; Cotten et al., Proc NatlAcad Sci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3):141-64,1994; Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992). Detailsconcerning the generation and use of rAAV vectors are described, forexample, in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporatedherein by reference in their entireties for all purposes.

In one embodiment, a retroviral expression vector can be used tointroduce a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) into a host cell for expression. These systems have beendescribed previously and are generally well known in the art (Mann etal., Cell, 33:153-159, 1983; Nicolas and Rubinstein, In: Vectors: Asurvey of molecular cloning vectors and their uses, Rodriguez andDenhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, In:Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188,1986). In a specific embodiment, the retroviral vector is a lentiviralvector (see, for example, Naldini et al., Science, 272(5259):263-267,1996; Zufferey et al., Nat Biotechnol, 15(9):871-875, 1997; Blomer etal., J Virol., 71(9):6641-6649, 1997; U.S. Pat. Nos. 6,013,516 and5,994,136).

Non-limiting examples of vectors for prokaryotic expression includeplasmids such as pRSET, pET, pBAD, etc., wherein the promoters used inprokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc.Examples of vectors for eukaryotic expression include: (i) forexpression in yeast, vectors such as pAO, pPIC, pYES, pMET, usingpromoters such as AOX1, GAP, GAL1, AUG1, etc; (ii) for expression ininsect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., usingpromoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc., and (iii)for expression in mammalian cells, vectors such as pSVL, pCMV, pRc/RSV,pcDNA3, pBPV, etc., and vectors derived form viral systems such asvaccinia virus, adeno-associated viruses, herpes viruses, retroviruses,etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, andβ-actin.

In certain embodiments, the cell-culture expression of ADAMTS13 maycomprise the use of a microcarrier. The present invention provides,among other aspect, methods of large-scale ADAMTS protein expression. Insome embodiments, the cell-cultures of the embodiments can be performedin large bioreactors under conditions suitable for providing highvolume-specific culture surface areas to achieve high cell densities andprotein expression. One means for providing such growth conditions is touse microcarriers for cell-culture in stirred tank bioreactors. Inanother embodiment, these growth requirements are met via the use of asuspension cell culture.

B. Cultivation Methods

In certain embodiments, ADAMTS13 expression can comprise the use of acell culture system operated under a batch or continuous mode ofoperation. For example, when batch cell cultures are utilized, they maybe operated under single batch, fed-batch, or repeated-batch mode.Likewise, continuous cell cultures may be operated under, for example,perfusion, turbidostat or chemostat mode. Batch and continuous cellcultivation may be performed under either suspension or adherenceconditions. When operated under suspension conditions, the cells will befreely suspended and mixed within the culture medium. Alternatively,under adherence conditions, the cells will be bound to a solid phase,for example, a microcarrier, a porous microcarrier, disk carrier,ceramic cartridge, hollow fiber, flat sheet, gel matrix, and the like.

A batch culture is typically a large scale cell culture in which a cellinoculum is cultured to a maximum density in a tank or fermenter, andharvested and processed as a single batch. A fed-batch culture ittypically a batch culture which is supplied with either fresh nutrients(e.g., growth-limiting substrates) or additives (e.g., precursors toproducts). The feed solution is usually highly concentrated to avoiddilution of the bioreactor. In a repeated-batch culture, the cells areplaced in a culture medium and grown to a desired cell density. To avoidthe onset of a decline phase and cell death, the culture is then dilutedwith complete growth medium before the cells reach their maximumconcentration. The amount and frequency of dilution varies widely anddepends on the growth characteristics of the cell line and convenienceof the culture process. The process can be repeated as many times asrequired and, unless cells and medium are discarded at subculture, thevolume of culture will increase stepwise as each dilution is made. Theincreasing volume may be handled by having a reactor of sufficient sizeto allow dilutions within the vessel or by dividing the diluted cultureinto several vessels. The rationale of this type of culture is tomaintain the cells in an exponentially growing state. Serial subcultureis characterized in that the volume of culture is always increasingstepwise, there can be multiple harvests, the cells continue to grow andthe process can continue for as long as desired. In certain embodiments,an ADAMTS protein (e.g., ADAMTS13) may be recovered after harvesting thesupernatant of a batch culture.

A continuous culture can be a suspension culture that is continuouslysupplied with nutrients by the inflow of fresh medium, wherein theculture volume is usually kept constant by the concomitant removal ofspent medium. In chemostat and turbidostat methods, the extracted mediumcontains cells. Thus, the cells remaining in the cell culture vesselmust grow to maintain a steady state. In the chemostat method, thegrowth rate is typically controlled by controlling the dilution rate,i.e., the rate at which fresh medium is added. The growth rate of thecells in the culture may be controlled, for example, at a sub-maximalgrowth rate, by alteration of the dilution rate. In contrast, in theturbidostat method, the dilution rate is set to permit the maximumgrowth rate that the cells can achieve at the given operatingconditions, such as pH and temperature.

In a perfusion culture, the extracted medium is depleted of cells, whichare retained in the culture vessel, for example, by filtration or bycentrifugal methods that lead to the reintroduction of the cells intothe culture. However, typically membranes used for filtration do notretain 100% of cells, and so a proportion are removed when the medium isextracted. It may not be crucial to operate perfusion cultures at veryhigh growth rates, as the majority of the cells are retained in theculture vessel.

Stirred-tank reactor system can be used for batch and continuous cellcultures operated under suspension or adherent modes. Generally, thestirred-tank reactor system can be operated as any conventionalstirred-tank reactor with any type of agitator such as a Rushton,hydrofoil, pitched blade, or marine.

C. Culture Mediums

ADAMTS13 may be expressed in culture mediums which are free ofexogenously added protein. “Protein free culture medium” and relatedterms refers to culture medium lacking protein that is from a sourceexogenous to or other than the cells in the culture, which naturallyshed proteins during growth. In one embodiment, an ADAMTS13 protein canbe expressed in a medium which is free of exogenously added protein(i.e., protein-free) and is supplemented with zinc, calcium, and/ornicotinamide (vitamin B3). In certain embodiments, the protein freeculture medium contains a polyamine. For example, at a concentration ofat least 2 mg/L, or at or about between 2 mg/L and 30 mg/L, or at orabout between 2 mg/L and 8 mg/L. In a specific embodiment, the polyamineis putrescine. Exemplary protein free culture mediums are taught in U.S.Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, U.S. PatentApplication Publication Numbers 2008/0009040 and 2007/0212770, and U.S.patent application Ser. No. 12/847,999, the disclosures of which areincorporated herein by reference in their entireties for all purposes.

Methods of preparing animal protein-free and chemically defined culturemediums are known in the art, for example in U.S. Pat. Nos. 6,171,825and 6,936,441, WO 2007/077217, and U.S. Patent Application PublicationNumbers 2008/0009040 and 2007/0212770, the disclosures of which areincorporated herein by reference in their entireties for all purposes.In one embodiment, the culture medium used to express an ADAMTS13protein is animal protein-free or oligopeptide-free medium. In certainembodiments, the culture medium may be chemically defined. In certainembodiments, the culture media may contain at least one polyamine at aconcentration of about 0.5 mg/L to about 10 mg/L.

ADAMTS13 protein can also be expressed in culture mediums which are freeof exogenously added oligopeptides. In one embodiment, ADAMTS13 isexpressed in a culture medium which is free of exogenously addedoligopeptides (i.e., polypeptide-free) and is supplemented with zinc,calcium, and/or nicotinamide (vitamin B3). In certain embodiments, theoligopeptide free culture medium contains a polyamine. For example, at aconcentration of at least 2 mg/L, or at or about between 2 mg/L and 30mg/L, or at or about between 2 mg/L and 8 mg/L. In a specificembodiment, the polyamine is putrescine. Exemplary oligopeptide freeculture mediums are taught in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO2007/077217, U.S. Patent Application Publication Numbers 2008/0009040and 2007/0212770, and U.S. patent application Ser. No. 12/847,999 thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

ADAMTS13 protein can also be expressed in culture mediums which are freeof serum. In one embodiment, ADAMTS13 is expressed in a culture mediumwhich is free of exogenously added serum (i.e., serum-free) and issupplemented with zinc, calcium, and/or nicotinamide (vitamin B3). Incertain embodiments, the serum-free culture medium contains a polyamine.For example, at a concentration of at least 2 mg/L, or at or aboutbetween 2 mg/L and 30 mg/L, or at or about between 2 mg/L and 8 mg/L. Ina specific embodiment, the polyamine is putrescine. Exemplary serum-freeculture mediums are taught in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO2007/077217, U.S. Patent Application Publication Numbers 2008/0009040and 2007/0212770, and U.S. patent application Ser. No. 12/847,999, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

ADAMTS13 protein can also be expressed in culture mediums which are freeof animal proteins. In one embodiment, ADAMTS13 is expressed in aculture medium which is free of exogenously added animal proteins orpolypeptides (i.e., animal protein free) and is supplemented with zinc,calcium, and/or nicotinamide (vitamin B3). In certain embodiments, theanimal protein free culture medium contains a polyamine. For example, ata concentration of at least 2 mg/L, or at or about between 2 mg/L and 30mg/L, or at or about between 2 mg/L and 8 mg/L. In a specificembodiment, the polyamine is putrescine. Exemplary animal protein freeculture mediums are taught in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO2007/077217, U.S. Patent Application Publication Numbers 2008/0009040and 2007/0212770, and U.S. patent application Ser. No. 12/847,999, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

ADAMTS13 proteins can also be expressed in culture mediums supplementedwith additional calcium, zinc, and/or vitamin B3, as described in U.S.patent application Ser. No. 12/847,999, the disclosure of which isincorporated herein by reference in its entirety for all purposes. Incertain embodiments, the medium may be an animal protein-free,oligopeptide-free, or chemically defined medium. In certain embodiments,the animal protein-free or oligopeptide free medium is prepared astaught in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, andU.S. Patent Application Publication Numbers 2008/0009040 and2007/0212770, the disclosures of which are incorporated herein byreference in their entireties for all purposes, both of which areincorporated herein by reference in their entireties for all purposes,and supplemented with additional calcium, zinc, and/or vitamin B3. In aspecific embodiment, the chemically defined culture medium may besimilar to a Dulbecco's Modified Eagle's Media (DMEM), which has beensupplemented with additional calcium, zinc, and/or vitamin B3, in orderto increase the specific activity of an ADAMTS protein expressed in acell cultured in the medium. In yet other embodiments, the culturemedium is animal component free. In another embodiment, the culturemedium contains protein, e.g., animal protein from serum such as fetalcalf serum. In another embodiment, the culture has recombinant proteinsexogenously added. In another embodiment, the proteins are from acertified pathogen free animal.

D. ADAMTS13 Purification

Methods for native ADAMTS13 purification from pooled plasma are wellknown in the art. Likewise, methods for the expression and purificationof recombinant ADAMTS13 are also known in the art. For example,recombinant expression and purification are taught by Plaimauer andScheiflinger, Semin Hematol. 2004 January; 41(1):24-33; Plaimauer B etal., F. Blood. 2002 Nov. 15; 100(10):3626-32. Epub 2002 Jul. 12; Bruno Ket al., J Thromb Haemost. 2005 May; 3(5):1064-73, and U.S. patentapplication Ser. No. 12/847,999, the disclosures of which are allexpressly incorporated by reference herein in their entireties for allpurposes.

Additionally, the present disclosure provides additional methods for thepurification of recombinant ADAMTS13. In one embodiment, recombinantADAMTS13 is expressed in recombinant CHO cells and purified from theresulting conditioned media by POROS HS cation exchange chromatography.To eliminate undesirable dimeric, monomeric, and/or aggregated ADAMTS13,the composition in then subjected to size exclusion gel filtration.ADAMTS13 compositions will generally also be subjected to at least one,preferably two, viral removal or inactivation steps.

In certain embodiments, the methods provided herein for the preparationof an ADAMTS13 formulation will further include at least one viralinactivation or removal steps. In certain embodiments, the methodsprovided herein will include at least two or at least three, viralinactivation or removal steps. Non-limiting examples of viralinactivation or removal steps that may be employed with the methodsprovided herein include, solvent detergent treatment (Horowitz et al.,Blood Coagul Fibrinolysis 1994 (5 Suppl 3):S21-S28 and Kreil et al.,Transfusion 2003 (43):1023-1028, the disclosures of which are expresslyincorporated by reference herein in their entireties for all purposes),nanofiltration (Hamamoto et al., Vox Sang 1989 (56)230-236 and Yuasa etal., J Gen Virol. 1991 (72 (pt 8)):2021-2024, the disclosures of whichare expressly incorporated by reference herein in their entireties forall purposes). In a preferred embodiment, the present invention providesmethods for the preparation of an ADAMTS13 formulation comprisingsolvent detergent treatment and nanofiltration.

In one embodiment, a method is provided for providing a virally safeADAMTS13 protein formulation, the method comprising the steps of (a)culturing a cell harboring a nucleic acid encoding an ADAMTS13 proteinin a culture medium; (b) recovering a portion of the culture mediumsupernatant containing the ADAMTS13 protein; (c) enriching the ADAMTS13protein with a chromatographic step; (d) performing at least one virusinactivation or removal step; and (e) formulating the enriched ADAMTS13composition according to a formulation provided herein, therebyproviding a virally safe ADAMTS13 formulation. In one embodiment, theculture medium contains zinc, calcium, and optionally nicotinamide(vitamin B3). In a preferred embodiment, the step of culturing a cellcomprises a continuous culture (e.g., perfusion or chemostatic culture).In another preferred embodiment, the culture is maintained at atemperature between 34° C. and 37° C. In yet another preferredembodiment, the culture is maintained at a pH between 6.9 and 7.2. Inone embodiment, the virus removal step is nanofiltration.

In aspect embodiment, the present invention provides a method formanufacturing a stabilized ADAMTS13 formulation, the method comprisingthe steps of: (a) expressing an ADAMTS13 protein, or a biologicallyactive derivative thereof, in a cell cultured in a medium comprisingzinc at a concentration from about 2 μM to about 12 μM and calcium at aconcentration from about 0.5 mM to about 1.5 mM; (b) purifying theADAMTS13 protein; and (c) preparing a formulation according to any oneof the formulations provided herein.

In one embodiment, the present invention provides a method formanufacturing a stabilized ADAMTS13 formulation, the method comprisingthe steps of: (i) expressing an ADAMTS13 protein, or a biologicallyactive derivative thereof, in a cell cultured in a medium comprisingzinc at a concentration from about 2 μM to about 12 μM and calcium at aconcentration from about 0.5 mM to about 1.5 mM; (ii) purifying theADAMTS13 protein; and (iii) preparing a formulation comprising (a) atleast 100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 200 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 1.0 mM and about 10.0 mM calcium. In a preferredembodiment, the formulation contains between about 2.0 and about 4.0 mMcalcium.

In another embodiment of the stabilized A13 formulation, the formulationcomprises between about 2% and about 6% of a sugar and/or sugar alcohol.In a preferred embodiment, the formulation comprises between about 3%and about 5% of a sugar and/or sugar alcohol. In a specific embodiment,the formulation comprises about 4% of a sugar and/or sugar alcohol. Inone embodiment, the sugar and/or sugar alcohol is selected from thegroup consisting of sucrose, trehalose, mannitol, and a combinationthereof. In a preferred embodiment, the sugar and/or sugar alcohol is amixture of sucrose and mannitol.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 0.01% and about 0.1% of a non-ionic surfactant.In a preferred embodiment, the formulation comprises about 0.05% of anon-ionic surfactant. In one embodiment, the surfactant is selected fromthe group consisting of Polysorbate 20, Polysorbate 80, Pluronic F-68,and BRIJ 35. In a preferred embodiment, the surfactant is Polysorbate80.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 5 mM and about 100 mM of a buffering agent. In apreferred embodiment, the formulation comprises between about 10 mM andabout 50 mM of a buffering agent. In another embodiment, the bufferingagent is histidine or HEPES. In a preferred embodiment, the bufferingagent is histidine. In one embodiment, the pH of the formulation isbetween about 6.5 and 7.5. In a preferred embodiment, the pH of theformulation is 7.0±0.2.

In one embodiment of the stabilized A13 formulation, the formulationfurther comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedformulation of ADAMTS13 (A13) comprising (a) at least 100 units ADAMTS13activity per mg ADAMTS13; (b) 0 to 200 mM NaCl; (c) 2 mM to 4 mMcalcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method formanufacturing a stabilized ADAMTS13 formulation, the method comprisingthe steps of: (i) expressing an ADAMTS13 protein, or a biologicallyactive derivative thereof, in a cell cultured in a medium comprisingzinc at a concentration from about 2 μM to about 12 μM and calcium at aconcentration from about 0.5 mM to about 1.5 mM; (ii) purifying theADAMTS13 protein; and (iii) preparing a formulation comprising (a) atleast 100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 100 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 1.0 mM and about 10.0 mM calcium. Ina preferred embodiment, the formulation contains between about 2.0 andabout 4.0 mM calcium.

In another embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 2% and about 6% of a sugar and/orsugar alcohol. In a preferred embodiment, the formulation comprisesbetween about 3% and about 5% of a sugar and/or sugar alcohol. In aspecific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 0.01% and about 0.1% of a non-ionicsurfactant. In a preferred embodiment, the formulation comprises about0.05% of a non-ionic surfactant. In one embodiment, the surfactant isselected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, and BRIJ 35. In a preferred embodiment, the surfactant isPolysorbate 80.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 5 mM and about 100 mM of a bufferingagent. In a preferred embodiment, the formulation comprises betweenabout 10 mM and about 50 mM of a buffering agent. In another embodiment,the buffering agent is histidine or HEPES. In a preferred embodiment,the buffering agent is histidine. In one embodiment, the pH of theformulation is between about 6.5 and 7.5. In a preferred embodiment, thepH of the formulation is 7.0±0.2.

In one embodiment of the stabilized low salt A13 formulation, theformulation further comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlow salt formulation of ADAMTS13 (A13) comprising (a) at least 100 unitsADAMTS13 activity per mg ADAMTS13; (b) 0 to 60 mM NaCl; (c) 2 mM to 4 mMcalcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method formanufacturing a stabilized ADAMTS13 formulation, the method comprisingthe steps of: (i) expressing an ADAMTS13 protein, or a biologicallyactive derivative thereof, in a cell cultured in a medium comprisingzinc at a concentration from about 2 μM to about 12 μM and calcium at aconcentration from about 0.5 mM to about 1.5 mM; (ii) purifying theADAMTS13 protein; and (iii) preparing a formulation comprising (a) atleast 100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 200 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 1.0 mM and about 10.0 mM calcium. Ina preferred embodiment, the formulation contains between about 2.0 andabout 4.0 mM calcium.

In another embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 2% and about 6% of a sugar and/orsugar alcohol. In a preferred embodiment, the formulation comprisesbetween about 3% and about 5% of a sugar and/or sugar alcohol. In aspecific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 0.01% and about 0.1% of a non-ionicsurfactant. In a preferred embodiment, the formulation comprises about0.05% of a non-ionic surfactant. In one embodiment, the surfactant isselected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, and BRIJ 35. In a preferred embodiment, the surfactant isPolysorbate 80.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 5 mM and about 100 mM of a bufferingagent. In a preferred embodiment, the formulation comprises betweenabout 10 mM and about 50 mM of a buffering agent. In another embodiment,the buffering agent is histidine or HEPES. In a preferred embodiment,the buffering agent is histidine. In one embodiment, the pH of theformulation is between about 6.5 and 7.5. In a preferred embodiment, thepH of the formulation is 7.0±0.2.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation further comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlyophilized formulation of ADAMTS13 (A13), wherein the formulation islyophilized from a liquid formulation comprising (a) at least 100 unitsADAMTS13 activity per mg ADAMTS13; (b) 0 to 200 mM NaCl; (c) 2 mM to 4mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method formanufacturing a stabilized ADAMTS13 formulation, the method comprisingthe steps of: (i) expressing an ADAMTS13 protein, or a biologicallyactive derivative thereof, in a cell cultured in a medium comprisingzinc at a concentration from about 2 μM to about 12 μM and calcium at aconcentration from about 0.5 mM to about 1.5 mM; (ii) purifying theADAMTS13 protein; and (iii) preparing a formulation comprising (a) atleast 100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 100 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 1.0 mM and about10.0 mM calcium. In a preferred embodiment, the formulation containsbetween about 2.0 and about 4.0 mM calcium.

In another embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 2% and about 6% ofa sugar and/or sugar alcohol. In a preferred embodiment, the formulationcomprises between about 3% and about 5% of a sugar and/or sugar alcohol.In a specific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 0.01% and about0.1% of a non-ionic surfactant. In a preferred embodiment, theformulation comprises about 0.05% of a non-ionic surfactant. In oneembodiment, the surfactant is selected from the group consisting ofPolysorbate 20, Polysorbate 80, Pluronic F-68, and BRIJ 35. In apreferred embodiment, the surfactant is Polysorbate 80.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 5 mM and about 100mM of a buffering agent. In a preferred embodiment, the formulationcomprises between about 10 mM and about 50 mM of a buffering agent. Inanother embodiment, the buffering agent is histidine or HEPES. In apreferred embodiment, the buffering agent is histidine. In oneembodiment, the pH of the formulation is between about 6.5 and 7.5. In apreferred embodiment, the pH of the formulation is 7.0±0.2.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation further comprises between about 0.5 μM and20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlow salt lyophilized formulation of ADAMTS13 (A13), wherein theformulation is lyophilized from a liquid formulation comprising (a) atleast 100 units ADAMTS13 activity per mg ADAMTS13; (b) 0 to 60 mM NaCl;(c) 2 mM to 4 mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose;(f) 0.025 to 0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH7.0±0.2).

1. Solvent and Detergent (S/D) Treatment

In order to inactivate various viral contaminants which may be presentin an ADAMTS13 formulation, one or more ADAMTS13 intermediate solutionsmay be subjected to a solvent detergent (S/D) treatment. Methods for thedetergent treatment of solutions are well known in the art (for reviewsee, Pelletier J P et al., Best Pract Res Clin Haematol. 2006;19(1):205-42, the disclosure of which is expressly incorporated byreference herein in its entirety for all purposes). Generally, anystandard S/D treatment may be used in conjunction with the methodsprovided herein. For example, an exemplary protocol for an S/D treatmentis provided below.

In one embodiment, Triton X-100, Tween-20, and tri(n-butyl)phosphate(TNBP) are added to an ADAMTS13 intermediate solution at finalconcentrations of at or about 1.0%, 0.3%, and 0.3%, respectively. Themixture is then stirred at a temperature at or about between 18° C. and25° C. for at least about an hour.

2. Nanofiltration and Ultra/Diafiltration

In order to reduce the viral load of an ADAMTS13 protein formulationprovided herein, the formulation, or an intermediate composition, may benanofiltered using a suitable nanofiltration device. In certainembodiments, the nanofiltration device will have a mean pore size of ator about between 15 nm and 200 nm. Examples of nanofilters suitable forthis use include, without limitation, DVD, DV 50, DV 20 (Pall),Viresolve NFP®, Viresolve NFR® (Millipore), Planova® 15N, 20N, 35N, and75N (Planova). In a specific embodiment, the nanofilter may have a meanpore size of at or about between 15 and 72 nm, or at or about between 19and 35 nm, or of at or about 15 nm, 19 nm, 20 nm, 35 nm, or 72 nm. In apreferred embodiment, the nanofilter will have a mean pore size of at orabout 19 nm, 20 nm, or 35 nm, such as an Asahi PLANOVA® 20N or PLANOVA®35N filter or equivalent thereof.

Subsequent to nanofiltration, the filtrate may optionally beconcentrated by ultrafiltration and/or the buffer composition adjustedby diafiltration. In certain embodiments, the ultrafiltration is carriedout in a cassette with an open channel screen and the ultrafiltrationmembrane has a nominal molecular weight cut off (NMWCO) of less than ator about 175 kDa or less than at or about 170, 160, 150, 140, 130, 120,110, 100, 90, 80, 70, 60, 50, 40, 30, or fewer kDa. In a preferredembodiment, the ultrafiltration membrane has a NMWCO of no more than 30kDa. In another preferred embodiment, the ultrafiltration membrane has aNMWCO of no more than 30 kDa.

3. Lyophilization and Heat Treatment

In yet other embodiments, the viral activity of a lyophilized ADAMTS13formulation, which may have previously been subjected to other viralinactivation or removal steps such as nanofiltration, may be furtherreduced by heat treatment of the lyophilized composition. Heattreatments for the inactivation of viral loads in blood factors are wellknown in the art (for example, see, Piszkiewicz et al., Thromb Res. 1987Jul. 15; 47(2):235-41; Piszkiewicz et al., Curr Stud Hematol BloodTransfus. 1989; (56):44-54; Epstein and Fricke, Arch Pathol Lab Med.1990 March; 114(3):335-40).

VI. Administration and Methods of Treatment

The formulations can be administered for therapeutic or prophylactictreatments. Generally, for therapeutic applications, formulations areadministered to a subject with a disease or condition associated withADAMTS13 or VWF dysfunction or otherwise in need thereof, in a“therapeutically effective dose.” Formulations and amounts effective forthese uses will depend upon the severity of the disease or condition andthe general state of the patient's health. Single or multipleadministrations of the formulations may be administered depending on thedosage and frequency as required and tolerated by the patient.

A “patient” or “subject” for the purposes of the present inventionincludes both humans and other animals, particularly mammals. Thus thecompositions, formulations, and methods are applicable to both humantherapy and veterinary applications. In a particular embodiment thepatient is a mammal, and in one embodiment, is a human. Other knowntreatments and therapies for conditions associated with ADAMTS13 or VWFdysfunction can be used in combination with the formulations and methodsprovided by the invention.

In one aspect of the invention, methods of making stabilized rA13formulation having high specific activities are provided. In oneembodiment, the method comprises the steps of expressing an ADAMTS13protein, or a biologically active derivative thereof, in a cell culturedin a medium comprising zinc at a concentration from about 2 μM to about12 μM and calcium at a concentration from about 0.5 mM to about 1.5 mM,purifying the A13 protein, and preparing a formulation as describedabove. In certain embodiments, the culture media is further supplementedwith vitamin B3 at a concentration from about 1 mg/L to about 20 mg/L.

In another aspect, the present invention provides methods of treating orpreventing a disease or condition associated with an ADAMTS13 or VWFdysfunction. In another embodiment, the invention provides methods oftreating or preventing a disease or condition associated with theformation and/or presence of one or more thrombus. In anotherembodiment, the invention provides methods of disintegrating one or morethrombus in a subject in need thereof. In yet other embodiments, theinvention provides methods of treating or preventing an infarction insubject in need thereof. Generally, the methods provided by theinvention comprise administering an ADAMTS13 formulation as providedherein to a subject in need thereof.

Non-limiting examples of disorders associated with the formation and/orthe presence of one or more thrombus are hereditary thromboticthrombocytopenic purpura (TTP), acquired TTP, arterial thrombosis, acutemyocardial infarction (AMI), stroke, sepsis, and disseminatedintravascular coagulation (DIC).

Non-limiting examples of disorders associated with an infarction,include without limitation, myocardial infarction (heart attack),pulmonary embolism, cerebrovascular events such as stroke, peripheralartery occlusive disease (such as gangrene), antiphospholipid syndrome,sepsis, giant-cell arteritis (GCA), hernia, and volvulus.

A. Treatment and Prophylaxis for ADAMTS13 and vWF Dysfunction

In aspect embodiment, the present invention provides a method fortreating or preventing a disease or condition associated with anADAMTS13 or VWF dysfunction, the method comprising administering to asubject in need thereof an ADAMTS13 formulation according to any one ofthe formulations provided herein.

In one embodiment, the present invention provides a method for treatingor preventing a disease or condition associated with an ADAMTS13 or VWFdysfunction, the method comprising administering to a subject in needthereof an ADAMTS13 formulation comprising (a) at least 100 unitsADAMTS13 activity (i.e., FRETS-vWF73 activity) per mg ADAMTS13; (b) 0 mMto 200 mM of a pharmaceutically acceptable salt; (c) 0.5 mM to 20 mMcalcium; (d) a sugar and/or sugar alcohol; (e) a nonionic surfactant;and (f) a buffering agent for maintaining a pH between 6.0 and 8.0. Inone embodiment, the stabilized formulation of ADAMTS13 comprises atleast 200 units A13 activity per mg ADAMTS13. In another embodiment, thestabilized formulation of ADAMTS13 comprises at least 400 units A13activity per mg ADAMTS13. In a preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 600 units A13 activity per mgADAMTS13. In a more preferred embodiment, the stabilized formulation ofADAMTS13 comprises at least 800 units A13 activity per mg ADAMTS13. Inyet another preferred embodiment, the stabilized formulation of ADAMTS13comprises at least 1000 units A13 activity per mg ADAMTS13. In oneembodiment, the stabilized formulation of ADAMTS13 comprises betweenabout 100 units and about 2000 units of ADAMTS13 activity per mgADAMTS13.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 1.0 mM and about 10.0 mM calcium. In a preferredembodiment, the formulation contains between about 2.0 and about 4.0 mMcalcium.

In another embodiment of the stabilized A13 formulation, the formulationcomprises between about 2% and about 6% of a sugar and/or sugar alcohol.In a preferred embodiment, the formulation comprises between about 3%and about 5% of a sugar and/or sugar alcohol. In a specific embodiment,the formulation comprises about 4% of a sugar and/or sugar alcohol. Inone embodiment, the sugar and/or sugar alcohol is selected from thegroup consisting of sucrose, trehalose, mannitol, and a combinationthereof. In a preferred embodiment, the sugar and/or sugar alcohol is amixture of sucrose and mannitol.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 0.01% and about 0.1% of a non-ionic surfactant.In a preferred embodiment, the formulation comprises about 0.05% of anon-ionic surfactant. In one embodiment, the surfactant is selected fromthe group consisting of Polysorbate 20, Polysorbate 80, Pluronic F-68,and BRIJ 35. In a preferred embodiment, the surfactant is Polysorbate80.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 5 mM and about 100 mM of a buffering agent. In apreferred embodiment, the formulation comprises between about 10 mM andabout 50 mM of a buffering agent. In another embodiment, the bufferingagent is histidine or HEPES. In a preferred embodiment, the bufferingagent is histidine. In one embodiment, the pH of the formulation isbetween about 6.5 and 7.5. In a preferred embodiment, the pH of theformulation is 7.0±0.2.

In one embodiment of the stabilized A13 formulation, the formulationfurther comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedformulation of ADAMTS13 (A13) comprising (a) at least 100 units ADAMTS13activity per mg ADAMTS13; (b) 0 to 200 mM NaCl; (c) 2 mM to 4 mMcalcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method for treatingor preventing a disease or condition associated with an ADAMTS13 or VWFdysfunction, the method comprising administering to a subject in needthereof an ADAMTS13 formulation comprising (a) at least 100 unitsADAMTS13 activity (i.e., FRETS-vWF73 activity) per mg ADAMTS13; (b) 0 mMto 100 mM of a pharmaceutically acceptable salt; (c) 0.5 mM to 20 mMcalcium; (d) a sugar and/or sugar alcohol; (e) a nonionic surfactant;and (f) a buffering agent for maintaining a pH between 6.0 and 8.0. Inone embodiment, the stabilized formulation of ADAMTS13 comprises atleast 200 units A13 activity per mg ADAMTS13. In another embodiment, thestabilized formulation of ADAMTS13 comprises at least 400 units A13activity per mg ADAMTS13. In a preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 600 units A13 activity per mgADAMTS13. In a more preferred embodiment, the stabilized formulation ofADAMTS13 comprises at least 800 units A13 activity per mg ADAMTS13. Inyet another preferred embodiment, the stabilized formulation of ADAMTS13comprises at least 1000 units A13 activity per mg ADAMTS13. In oneembodiment, the stabilized formulation of ADAMTS13 comprises betweenabout 100 units and about 2000 units of ADAMTS13 activity per mgADAMTS13.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 1.0 mM and about 10.0 mM calcium. Ina preferred embodiment, the formulation contains between about 2.0 andabout 4.0 mM calcium.

In another embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 2% and about 6% of a sugar and/orsugar alcohol. In a preferred embodiment, the formulation comprisesbetween about 3% and about 5% of a sugar and/or sugar alcohol. In aspecific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 0.01% and about 0.1% of a non-ionicsurfactant. In a preferred embodiment, the formulation comprises about0.05% of a non-ionic surfactant. In one embodiment, the surfactant isselected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, and BRIJ 35. In a preferred embodiment, the surfactant isPolysorbate 80.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 5 mM and about 100 mM of a bufferingagent. In a preferred embodiment, the formulation comprises betweenabout 10 mM and about 50 mM of a buffering agent. In another embodiment,the buffering agent is histidine or HEPES. In a preferred embodiment,the buffering agent is histidine. In one embodiment, the pH of theformulation is between about 6.5 and 7.5. In a preferred embodiment, thepH of the formulation is 7.0±0.2.

In one embodiment of the stabilized low salt A13 formulation, theformulation further comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlow salt formulation of ADAMTS13 (A13) comprising (a) at least 100 unitsADAMTS13 activity per mg ADAMTS13; (b) 0 to 60 mM NaCl; (c) 2 mM to 4 mMcalcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method for treatingor preventing a disease or condition associated with an ADAMTS13 or VWFdysfunction, the method comprising administering to a subject in needthereof an ADAMTS13 formulation comprising (a) at least 100 unitsADAMTS13 activity (i.e., FRETS-vWF73 activity) per mg ADAMTS13; (b) 0 mMto 200 mM of a pharmaceutically acceptable salt; (c) 0.5 mM to 20 mMcalcium; (d) a sugar and/or sugar alcohol; (e) a nonionic surfactant;and (f) a buffering agent for maintaining a pH between 6.0 and 8.0. Inone embodiment, the stabilized formulation of ADAMTS13 comprises atleast 200 units A13 activity per mg ADAMTS13. In another embodiment, thestabilized formulation of ADAMTS13 comprises at least 400 units A13activity per mg ADAMTS13. In a preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 600 units A13 activity per mgADAMTS13. In a more preferred embodiment, the stabilized formulation ofADAMTS13 comprises at least 800 units A13 activity per mg ADAMTS13. Inyet another preferred embodiment, the stabilized formulation of ADAMTS13comprises at least 1000 units A13 activity per mg ADAMTS13. In oneembodiment, the stabilized formulation of ADAMTS13 comprises betweenabout 100 units and about 2000 units of ADAMTS13 activity per mgADAMTS13.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 1.0 mM and about 10.0 mM calcium. Ina preferred embodiment, the formulation contains between about 2.0 andabout 4.0 mM calcium.

In another embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 2% and about 6% of a sugar and/orsugar alcohol. In a preferred embodiment, the formulation comprisesbetween about 3% and about 5% of a sugar and/or sugar alcohol. In aspecific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 0.01% and about 0.1% of a non-ionicsurfactant. In a preferred embodiment, the formulation comprises about0.05% of a non-ionic surfactant. In one embodiment, the surfactant isselected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, and BRIJ 35. In a preferred embodiment, the surfactant isPolysorbate 80.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 5 mM and about 100 mM of a bufferingagent. In a preferred embodiment, the formulation comprises betweenabout 10 mM and about 50 mM of a buffering agent. In another embodiment,the buffering agent is histidine or HEPES. In a preferred embodiment,the buffering agent is histidine. In one embodiment, the pH of theformulation is between about 6.5 and 7.5. In a preferred embodiment, thepH of the formulation is 7.0±0.2.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation further comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlyophilized formulation of ADAMTS13 (A13), wherein the formulation islyophilized from a liquid formulation comprising (a) at least 100 unitsADAMTS13 activity per mg ADAMTS13; (b) 0 to 200 mM NaCl; (c) 2 mM to 4mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method for treatingor preventing a disease or condition associated with an ADAMTS13 or VWFdysfunction, the method comprising administering to a subject in needthereof an ADAMTS13 formulation comprising (a) at least 100 unitsADAMTS13 activity (i.e., FRETS-vWF73 activity) per mg ADAMTS13; (b) 0 mMto 100 mM of a pharmaceutically acceptable salt; (c) 0.5 mM to 20 mMcalcium; (d) a sugar and/or sugar alcohol; (e) a nonionic surfactant;and (f) a buffering agent for maintaining a pH between 6.0 and 8.0. Inone embodiment, the stabilized formulation of ADAMTS13 comprises atleast 200 units A13 activity per mg ADAMTS13. In another embodiment, thestabilized formulation of ADAMTS13 comprises at least 400 units A13activity per mg ADAMTS13. In a preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 600 units A13 activity per mgADAMTS13. In a more preferred embodiment, the stabilized formulation ofADAMTS13 comprises at least 800 units A13 activity per mg ADAMTS13. Inyet another preferred embodiment, the stabilized formulation of ADAMTS13comprises at least 1000 units A13 activity per mg ADAMTS13. In oneembodiment, the stabilized formulation of ADAMTS13 comprises betweenabout 100 units and about 2000 units of ADAMTS13 activity per mgADAMTS13.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 1.0 mM and about10.0 mM calcium. In a preferred embodiment, the formulation containsbetween about 2.0 and about 4.0 mM calcium.

In another embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 2% and about 6% ofa sugar and/or sugar alcohol. In a preferred embodiment, the formulationcomprises between about 3% and about 5% of a sugar and/or sugar alcohol.In a specific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 0.01% and about0.1% of a non-ionic surfactant. In a preferred embodiment, theformulation comprises about 0.05% of a non-ionic surfactant. In oneembodiment, the surfactant is selected from the group consisting ofPolysorbate 20, Polysorbate 80, Pluronic F-68, and BRIJ 35. In apreferred embodiment, the surfactant is Polysorbate 80.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 5 mM and about 100mM of a buffering agent. In a preferred embodiment, the formulationcomprises between about 10 mM and about 50 mM of a buffering agent. Inanother embodiment, the buffering agent is histidine or HEPES. In apreferred embodiment, the buffering agent is histidine. In oneembodiment, the pH of the formulation is between about 6.5 and 7.5. In apreferred embodiment, the pH of the formulation is 7.0±0.2.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation further comprises between about 0.5 μM and20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlow salt lyophilized formulation of ADAMTS13 (A13), wherein theformulation is lyophilized from a liquid formulation comprising (a) atleast 100 units ADAMTS13 activity per mg ADAMTS13; (b) 0 to 60 mM NaCl;(c) 2 mM to 4 mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose;(f) 0.025 to 0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH7.0±0.2).

B. Treatment and Prophylaxis for Thrombotic Diseases and Conditions

In aspect embodiment, the present invention provides a method fortreating or preventing a disease or condition with the formation and/orpresence of a thrombus, the method comprising administering to a subjectin need thereof an ADAMTS13 formulation according to any one of theformulations provided herein.

In one embodiment, the present invention provides a method for treatingor preventing a disease or condition associated with the formationand/or presence of a thrombus, the method comprising administering to asubject in need thereof an ADAMTS13 formulation comprising (a) at least100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 200 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 1.0 mM and about 10.0 mM calcium. In a preferredembodiment, the formulation contains between about 2.0 and about 4.0 mMcalcium.

In another embodiment of the stabilized A13 formulation, the formulationcomprises between about 2% and about 6% of a sugar and/or sugar alcohol.In a preferred embodiment, the formulation comprises between about 3%and about 5% of a sugar and/or sugar alcohol. In a specific embodiment,the formulation comprises about 4% of a sugar and/or sugar alcohol. Inone embodiment, the sugar and/or sugar alcohol is selected from thegroup consisting of sucrose, trehalose, mannitol, and a combinationthereof. In a preferred embodiment, the sugar and/or sugar alcohol is amixture of sucrose and mannitol.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 0.01% and about 0.1% of a non-ionic surfactant.In a preferred embodiment, the formulation comprises about 0.05% of anon-ionic surfactant. In one embodiment, the surfactant is selected fromthe group consisting of Polysorbate 20, Polysorbate 80, Pluronic F-68,and BRIJ 35. In a preferred embodiment, the surfactant is Polysorbate80.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 5 mM and about 100 mM of a buffering agent. In apreferred embodiment, the formulation comprises between about 10 mM andabout 50 mM of a buffering agent. In another embodiment, the bufferingagent is histidine or HEPES. In a preferred embodiment, the bufferingagent is histidine. In one embodiment, the pH of the formulation isbetween about 6.5 and 7.5. In a preferred embodiment, the pH of theformulation is 7.0±0.2.

In one embodiment of the stabilized A13 formulation, the formulationfurther comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedformulation of ADAMTS13 (A13) comprising (a) at least 100 units ADAMTS13activity per mg ADAMTS13; (b) 0 to 200 mM NaCl; (c) 2 mM to 4 mMcalcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method for treatingor preventing a disease or condition associated with the formationand/or presence of a thrombus, the method comprising administering to asubject in need thereof an ADAMTS13 formulation comprising (a) at least100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 100 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 1.0 mM and about 10.0 mM calcium. Ina preferred embodiment, the formulation contains between about 2.0 andabout 4.0 mM calcium.

In another embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 2% and about 6% of a sugar and/orsugar alcohol. In a preferred embodiment, the formulation comprisesbetween about 3% and about 5% of a sugar and/or sugar alcohol. In aspecific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 0.01% and about 0.1% of a non-ionicsurfactant. In a preferred embodiment, the formulation comprises about0.05% of a non-ionic surfactant. In one embodiment, the surfactant isselected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, and BRIJ 35. In a preferred embodiment, the surfactant isPolysorbate 80.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 5 mM and about 100 mM of a bufferingagent. In a preferred embodiment, the formulation comprises betweenabout 10 mM and about 50 mM of a buffering agent. In another embodiment,the buffering agent is histidine or HEPES. In a preferred embodiment,the buffering agent is histidine. In one embodiment, the pH of theformulation is between about 6.5 and 7.5. In a preferred embodiment, thepH of the formulation is 7.0±0.2.

In one embodiment of the stabilized low salt A13 formulation, theformulation further comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlow salt formulation of ADAMTS13 (A13) comprising (a) at least 100 unitsADAMTS13 activity per mg ADAMTS13; (b) 0 to 60 mM NaCl; (c) 2 mM to 4 mMcalcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method for treatingor preventing a disease or condition associated with the formationand/or presence of a thrombus, the method comprising administering to asubject in need thereof an ADAMTS13 formulation comprising (a) at least100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 200 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 1.0 mM and about 10.0 mM calcium. Ina preferred embodiment, the formulation contains between about 2.0 andabout 4.0 mM calcium.

In another embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 2% and about 6% of a sugar and/orsugar alcohol. In a preferred embodiment, the formulation comprisesbetween about 3% and about 5% of a sugar and/or sugar alcohol. In aspecific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 0.01% and about 0.1% of a non-ionicsurfactant. In a preferred embodiment, the formulation comprises about0.05% of a non-ionic surfactant. In one embodiment, the surfactant isselected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, and BRIJ 35. In a preferred embodiment, the surfactant isPolysorbate 80.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 5 mM and about 100 mM of a bufferingagent. In a preferred embodiment, the formulation comprises betweenabout 10 mM and about 50 mM of a buffering agent. In another embodiment,the buffering agent is histidine or HEPES. In a preferred embodiment,the buffering agent is histidine. In one embodiment, the pH of theformulation is between about 6.5 and 7.5. In a preferred embodiment, thepH of the formulation is 7.0±0.2.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation further comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlyophilized formulation of ADAMTS13 (A13), wherein the formulation islyophilized from a liquid formulation comprising (a) at least 100 unitsADAMTS13 activity per mg ADAMTS13; (b) 0 to 200 mM NaCl; (c) 2 mM to 4mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method for treatingor preventing a disease or condition associated with the formationand/or presence of a thrombus, the method comprising administering to asubject in need thereof an ADAMTS13 formulation comprising (a) at least100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 100 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 1.0 mM and about10.0 mM calcium. In a preferred embodiment, the formulation containsbetween about 2.0 and about 4.0 mM calcium.

In another embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 2% and about 6% ofa sugar and/or sugar alcohol. In a preferred embodiment, the formulationcomprises between about 3% and about 5% of a sugar and/or sugar alcohol.In a specific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 0.01% and about0.1% of a non-ionic surfactant. In a preferred embodiment, theformulation comprises about 0.05% of a non-ionic surfactant. In oneembodiment, the surfactant is selected from the group consisting ofPolysorbate 20, Polysorbate 80, Pluronic F-68, and BRIJ 35. In apreferred embodiment, the surfactant is Polysorbate 80.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 5 mM and about 100mM of a buffering agent. In a preferred embodiment, the formulationcomprises between about 10 mM and about 50 mM of a buffering agent. Inanother embodiment, the buffering agent is histidine or HEPES. In apreferred embodiment, the buffering agent is histidine. In oneembodiment, the pH of the formulation is between about 6.5 and 7.5. In apreferred embodiment, the pH of the formulation is 7.0±0.2.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation further comprises between about 0.5 μM and20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlow salt lyophilized formulation of ADAMTS13 (A13), wherein theformulation is lyophilized from a liquid formulation comprising (a) atleast 100 units ADAMTS13 activity per mg ADAMTS13; (b) 0 to 60 mM NaCl;(c) 2 mM to 4 mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose;(f) 0.025 to 0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH7.0±0.2).

C. Treatment and Prophylaxis for an Infarction

In aspect embodiment, the present invention provides a method fortreating or preventing an infarction, the method comprisingadministering to a subject in need thereof an ADAMTS13 formulationaccording to any one of the formulations provided herein.

In one embodiment, the present invention provides a method for treatingor preventing an infarction, the method comprising administering to asubject in need thereof an ADAMTS13 formulation comprising (a) at least100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 200 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 1.0 mM and about 10.0 mM calcium. In a preferredembodiment, the formulation contains between about 2.0 and about 4.0 mMcalcium.

In another embodiment of the stabilized A13 formulation, the formulationcomprises between about 2% and about 6% of a sugar and/or sugar alcohol.In a preferred embodiment, the formulation comprises between about 3%and about 5% of a sugar and/or sugar alcohol. In a specific embodiment,the formulation comprises about 4% of a sugar and/or sugar alcohol. Inone embodiment, the sugar and/or sugar alcohol is selected from thegroup consisting of sucrose, trehalose, mannitol, and a combinationthereof. In a preferred embodiment, the sugar and/or sugar alcohol is amixture of sucrose and mannitol.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 0.01% and about 0.1% of a non-ionic surfactant.In a preferred embodiment, the formulation comprises about 0.05% of anon-ionic surfactant. In one embodiment, the surfactant is selected fromthe group consisting of Polysorbate 20, Polysorbate 80, Pluronic F-68,and BRIJ 35. In a preferred embodiment, the surfactant is Polysorbate80.

In one embodiment of the stabilized A13 formulation, the formulationcomprises between about 5 mM and about 100 mM of a buffering agent. In apreferred embodiment, the formulation comprises between about 10 mM andabout 50 mM of a buffering agent. In another embodiment, the bufferingagent is histidine or HEPES. In a preferred embodiment, the bufferingagent is histidine. In one embodiment, the pH of the formulation isbetween about 6.5 and 7.5. In a preferred embodiment, the pH of theformulation is 7.0±0.2.

In one embodiment of the stabilized A13 formulation, the formulationfurther comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedformulation of ADAMTS13 (A13) comprising (a) at least 100 units ADAMTS13activity per mg ADAMTS13; (b) 0 to 200 mM NaCl; (c) 2 mM to 4 mMcalcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method for treatingor preventing an infarction, the method comprising administering to asubject in need thereof an ADAMTS13 formulation comprising (a) at least100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 100 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 1.0 mM and about 10.0 mM calcium. Ina preferred embodiment, the formulation contains between about 2.0 andabout 4.0 mM calcium.

In another embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 2% and about 6% of a sugar and/orsugar alcohol. In a preferred embodiment, the formulation comprisesbetween about 3% and about 5% of a sugar and/or sugar alcohol. In aspecific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 0.01% and about 0.1% of a non-ionicsurfactant. In a preferred embodiment, the formulation comprises about0.05% of a non-ionic surfactant. In one embodiment, the surfactant isselected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, and BRIJ 35. In a preferred embodiment, the surfactant isPolysorbate 80.

In one embodiment of the stabilized low salt A13 formulation, theformulation comprises between about 5 mM and about 100 mM of a bufferingagent. In a preferred embodiment, the formulation comprises betweenabout 10 mM and about 50 mM of a buffering agent. In another embodiment,the buffering agent is histidine or HEPES. In a preferred embodiment,the buffering agent is histidine. In one embodiment, the pH of theformulation is between about 6.5 and 7.5. In a preferred embodiment, thepH of the formulation is 7.0±0.2.

In one embodiment of the stabilized low salt A13 formulation, theformulation further comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlow salt formulation of ADAMTS13 (A13) comprising (a) at least 100 unitsADAMTS13 activity per mg ADAMTS13; (b) 0 to 60 mM NaCl; (c) 2 mM to 4 mMcalcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method for treatingor preventing an infarction, the method comprising administering to asubject in need thereof an ADAMTS13 formulation comprising (a) at least100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 200 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 1.0 mM and about 10.0 mM calcium. Ina preferred embodiment, the formulation contains between about 2.0 andabout 4.0 mM calcium.

In another embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 2% and about 6% of a sugar and/orsugar alcohol. In a preferred embodiment, the formulation comprisesbetween about 3% and about 5% of a sugar and/or sugar alcohol. In aspecific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 0.01% and about 0.1% of a non-ionicsurfactant. In a preferred embodiment, the formulation comprises about0.05% of a non-ionic surfactant. In one embodiment, the surfactant isselected from the group consisting of Polysorbate 20, Polysorbate 80,Pluronic F-68, and BRIJ 35. In a preferred embodiment, the surfactant isPolysorbate 80.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation comprises between about 5 mM and about 100 mM of a bufferingagent. In a preferred embodiment, the formulation comprises betweenabout 10 mM and about 50 mM of a buffering agent. In another embodiment,the buffering agent is histidine or HEPES. In a preferred embodiment,the buffering agent is histidine. In one embodiment, the pH of theformulation is between about 6.5 and 7.5. In a preferred embodiment, thepH of the formulation is 7.0±0.2.

In one embodiment of the stabilized lyophilized A13 formulation, theformulation further comprises between about 0.5 μM and 20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlyophilized formulation of ADAMTS13 (A13), wherein the formulation islyophilized from a liquid formulation comprising (a) at least 100 unitsADAMTS13 activity per mg ADAMTS13; (b) 0 to 200 mM NaCl; (c) 2 mM to 4mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f) 0.025 to0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH 7.0±0.2).

In one embodiment, the present invention provides a method for treatingor preventing an infarction, the method comprising administering to asubject in need thereof an ADAMTS13 formulation comprising (a) at least100 units ADAMTS13 activity (i.e., FRETS-vWF73 activity) per mgADAMTS13; (b) 0 mM to 100 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0. In one embodiment, the stabilized formulation ofADAMTS13 comprises at least 200 units A13 activity per mg ADAMTS13. Inanother embodiment, the stabilized formulation of ADAMTS13 comprises atleast 400 units A13 activity per mg ADAMTS13. In a preferred embodiment,the stabilized formulation of ADAMTS13 comprises at least 600 units A13activity per mg ADAMTS13. In a more preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 800 units A13 activity per mgADAMTS13. In yet another preferred embodiment, the stabilizedformulation of ADAMTS13 comprises at least 1000 units A13 activity permg ADAMTS13. In one embodiment, the stabilized formulation of ADAMTS13comprises between about 100 units and about 2000 units of ADAMTS13activity per mg ADAMTS13.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 1.0 mM and about10.0 mM calcium. In a preferred embodiment, the formulation containsbetween about 2.0 and about 4.0 mM calcium.

In another embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 2% and about 6% ofa sugar and/or sugar alcohol. In a preferred embodiment, the formulationcomprises between about 3% and about 5% of a sugar and/or sugar alcohol.In a specific embodiment, the formulation comprises about 4% of a sugarand/or sugar alcohol. In one embodiment, the sugar and/or sugar alcoholis selected from the group consisting of sucrose, trehalose, mannitol,and a combination thereof. In a preferred embodiment, the sugar and/orsugar alcohol is a mixture of sucrose and mannitol.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 0.01% and about0.1% of a non-ionic surfactant. In a preferred embodiment, theformulation comprises about 0.05% of a non-ionic surfactant. In oneembodiment, the surfactant is selected from the group consisting ofPolysorbate 20, Polysorbate 80, Pluronic F-68, and BRIJ 35. In apreferred embodiment, the surfactant is Polysorbate 80.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation comprises between about 5 mM and about 100mM of a buffering agent. In a preferred embodiment, the formulationcomprises between about 10 mM and about 50 mM of a buffering agent. Inanother embodiment, the buffering agent is histidine or HEPES. In apreferred embodiment, the buffering agent is histidine. In oneembodiment, the pH of the formulation is between about 6.5 and 7.5. In apreferred embodiment, the pH of the formulation is 7.0±0.2.

In one embodiment of the stabilized low salt lyophilized A13formulation, the formulation further comprises between about 0.5 μM and20 μM zinc.

In a specific embodiment, the present invention provides a stabilizedlow salt lyophilized formulation of ADAMTS13 (A13), wherein theformulation is lyophilized from a liquid formulation comprising (a) atleast 100 units ADAMTS13 activity per mg ADAMTS13; (b) 0 to 60 mM NaCl;(c) 2 mM to 4 mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose;(f) 0.025 to 0.1% Polysorbate 80; and (g) 10 mM to 50 mM histidine (pH7.0±0.2).

VII. ADAMTS13 Kits

In another aspect of the invention, kits are provided for the treatmentof a disease or condition associated with ADAMTS13 or VWF dysfunction.In one embodiment, the kit comprises a formulation of A13 or rA13, asprovided above. In some embodiments, the kits provided herein maycontain one or more dose of a liquid or lyophilized formulation asprovided herein. When the kits comprise a lyophilized A13 or rA13formulation, generally the kits will also contain a suitable liquid forreconstitution of the liquid formulation, for example, sterile water ora pharmaceutically acceptable buffer. In some embodiments, the kits maycomprise an ADAMTS13 formulation prepackaged in a syringe foradministration by a health care professional or for home use.

In one embodiment, a kit is provided comprising between about 10 unitsof FRETS-VWF73 activity and about 10,000 units of FRETS-VWF73 activity.In other embodiments, the kit may provide, for example, between about 20units of FRETS-VWF73 (U_(FV73)) activity and about 8,000 units ofFRETS-VWF73 activity, or between about 30 U_(FV73) and about 6,000U_(FV73), or between about 40 U_(FV73) and about 4,000 U_(FV73), orbetween about 50 U_(FV73) and about 3,000 U_(FV73), or between about 75U_(FV73) and about 2,500 U_(FV73), or between about 100 U_(FV73) andabout 2,000 U_(FV73), or between about 200 U_(FV73) and about 1,500U_(FV73), or between about other ranges therein. In certain embodiments,a kit may provide about 10 units of FRETS-VWF73 activity, or about 20,30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600,2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600,3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600,4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600,5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600,6,700, 6,800, 6,900, 7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600,7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600,8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600,9,700, 9,800, 9,900, 10,000 or more units of FRETS-VWF73 activity.

In certain embodiments, the kit will be for a single administration ordose of ADAMTS13. In other embodiments, the kit may contain multipledoses of ADAMTS13 for administration. In one embodiment, the kit maycomprise an ADAMTS13 formulation prepackaged in a syringe foradministration by a health care professional or for home use.

VIII. Examples A. Example 1 Expression of Recombinant ADAMTS13 (rA13)

A Chemostat cell-culture of the recombinant CHO cell line #640-2expressing human ADAMTS13, was grown in chemically defined BACD-A13medium supplemented with additional zinc and vitamin B3. The 10 Lculture was maintained for 53 days and rA13 protein and activityproduction was monitored over time.

Recombinant CHO cells expressing human ADAMTS13 were adapted to achemically defined proprietary medium (BCS medium). A DWCB was thawedand cell inoculum was prepared in BCS medium. Cells propagated from therA13 expression clone #640-2 were transferred to a 10 L bioreactor withRushton type impellers and cultivated in repeated batch cultures withproprietary BACD-A13 medium under an inline controlled pH of 7.15-7.20at 37° C. with a dissolved oxygen concentration of 20% air saturation.After 2 batch cultures were grown to the final working volume of 10 L,the bioreactor was switched to continuous medium feed on day 5 andoperated for an additional 48 days in a chemostat mode.

Samples of the supernatant from the bioreactors were taken weekly andanalyzed for rA13 protein production by ELISA and rA13 activity byFRETS-VWF73 assay. Cell counts were determined by Nucleocountertechnology. Dilution rates were measured and used for calculation ofgrowth rates and volumetric productivities.

Under continuous culture conditions using chemically defined BACD-A13medium supplemented with zinc and nicotinamide at a final concentrationof 1.432 mg/L ZnSO₄7H₂O and 7.02 mg/L nicotinamide, high levels of rA13protein production, between 0.9 and 1.3 mg/L/D, and specific activities,between about 800 and 1100 mU/μg rA13, were achieved (Table 1). Notably,volumetric and cell specific productivities increased over time in thelong term culture, likely due to increasing growth and dilution ratesover time. The high specific activity of the expressed rA13 could be atleast maintained at a constantly high level over at least entire 7 weeksthe culture was grown under chemostatic conditions. In fact, thespecific activity of the rA13 produced in the culture actually increasedfrom about 800 mU/μg A13 at week 2 to about 1100 mU/μg A13 at week 7.

TABLE 1 Fermentation data for batch experiment CP_07/18_M07: hA13 CHOKlon #985/1 985 DWCB #01. Specific Chemostat Cell Growth Dilution A13A13 Specific FRETS A13 Culture Concentration Rate Rate FRETS ELISAActivity Yield Yield Week No. [10⁶ Cells/ml] [1/d] [1/d] [mU/ml] [g/ml][mU/μg] [U/L/d] [mg/L/d] 2 1.43 0.36 0.36 1954 2.48 788 713 0.91 3 1.560.41 0.40 2254 2.32 972 913 0.94 4 1.46 0.38 0.40 2244 2.41 931 889 0.955 1.58 0.43 0.43 2514 2.88 873 1086 1.24 6 1.70 0.51 0.46 2737 2.71 10101270 1.26 7 1.76 0.53 0.52 2322 2.18 1065 1200 1.13

B. Example 2 Formulation of Purified Recombinant ADAMTS13 (rA13)

Recombinant ADAMTS13 was expressed in recombinant CHO cells and purifiedby anion exchange chromatography. The purified rA13 had a finalconcentration of approximately 750 μg/ml with a specific activity ofapproximately 850 mU/μg. rA13 was formulated in buffer containing 150 mMNaCl, 2% sucrose, 0.05% polysorbate 80, at a pH of 7.0 with 20 mM of abuffering agent selected from (1) histidine, (2) phosphate buffer, or(3) sodium citrate. Samples were then divided evenly and half thesamples were lyophilized.

Lyophilized samples were reconstituted with sterile water to a finalvolume equal to that of the pre-lyophilized formulation. A singlealiquot of each liquid and lyophilized formulation was thencharacterized by gel filtration by loading the sample onto a Superose 6GL column (GE Healthcare). As can be seen in FIG. 3, all of theformulations resulted in ADAMTS13 samples that ran as a single peakcorresponding to monomeric rA13 protein by gel filtration.

C. Example 3 Characterization of Activity Retention of rA13 FormulationsStored at 4° C. and 37° C.

rA13 samples formulated and aliquoted as in example 2 were stored ateither 4° C. or 37° C. for up to 6 months time. Solution formulationswere analyzed for rA13 protein concentration, as determined by ELISA,and rA13 activity by FRETS-VWF73 assay at 0, 1, 2, 3, 12, and 24 weeks(FIG. 1). Lyophilized samples were reconstituted with sterile water to afinal volume equal to that of the pre-lyophilized formulation andanalyzed similarly at 0, 2, 4, 8, 12, and 24 weeks (FIG. 2).

rA13 liquid formulations stored at 4° C. showed no loss in eitherantigen content, i.e. protein concentration, or FRETS-VWF73 activity attime points out to 6 months. As seen in FIG. 1, this was the case forall three formulations, buffered with histidine, phosphate, and sodiumcitrate, respectively. Conversely, liquid formulations, buffered withhistidine or sodium citrate, displayed nearly complete loss of activityafter one week at 37° C. (FIG. 1A; ⋄=histidine, □=sodium citrate). Thisloss in activity was accompanied with a spike in the antigen content atone week for these samples, suggesting that the rA13 proteins were beingdenatured. The histidine liquid formulation of rA13 displayeddrastically reduced antigen content at 3 and 6 months, suggesting thatthe protein was being degraded. Notably, liquid rA13 formulated withphosphate buffer showed significant activity at three weeks when storedat 37° C. (FIG. 1A; ◯=phosphate buffer). Consistently, the antigencontent of this formulation increased at a slower rate, suggesting thatrA13 denaturation is retarded when formulated with phosphate buffer.

rA13 lyophilized formulations appeared to be stable for at least 6months when stored at either 4° C. or 37° C. (FIG. 2). Lyophilized rA13formulated with phosphate buffer displayed a gradual loss of activity(30% at 6 months) when stored at 37° C., but not at 4° C., however, thisloss of activity was not accompanied by an increase in antigenavailability by ELISA (FIG. 2; ◯=phosphate buffer). No other lyophilizedformulation showed a loss of activity or increase in antigenavailability when stored at 4° C. or 37° C.

D. Example 4 Conformational Stability of rA13 in Formulations Stored at4° C. and 37° C.

rA13 samples formulated and aliquoted as in example 2 were stored ateither 4° C. or 37° C. for up to 6 months time. The global conformationsof rA13 formulated and stored in solution and lyophilized states wascharacterized by gel filtration at the time points described in example3. Results of gel filtration experiments performed with the histidineformulated rA13 at 0, 1, 2, 3, and 24 weeks for the liquid formulations(FIG. 4) and 0, 2, 4, 8, 12, and 24 weeks for the lyophilizedformulations (FIG. 5) are shown.

Liquid formulations with histidine, stored at 4° C. for up to 6 months,ran primarily as a monomer over gel filtration (FIG. 4A), suggestingthat the protein is very stable in solution at 4° C. This result isconsistent with those described in example 3, which showed that rA13formulated with histidine and stored in solution at 4° C. for up to 6months did not lose any FRETS-VWF73 activity. Conversely, rA13formulated with histidine and stored at 37° C., ran as higherorder/partially or wholly denatured species, as indicated by the reducedelution volumes/retention times (FIG. 4B). Consistent with the resultsseen in example 3, histidine formulated A13 stored at 37° C. for 6months appeared to be mostly degraded.

Gel filtration experiments were repeated for each formulation, stored at4° C. and 37° C., and relative areas under the eluted peaks wereintegrated in order to determine the relative populations of monomerrA13 (3), dimer rA13 (2), and aggregated/denatured rA13 (1) in theformulations. Results of the gel filtration experiments performed on thethree lyophilized formulations stored at 4° C. and 37° C. for six monthsare given in Table 2. Lyophilized samples were reconstituted withsterile water to a final volume equal to that of the pre-lyophilizedformulation prior to analysis via gel filtration.

TABLE 2 Relative area under the curve (AUC) for gel filtrations ofreconstituted ADAMTS13 lyophilized formulations. Relative peak area [%]Lyo Lyo 6 m Lyo 6 m Peak No start 4° C. 37° C. Lyo_His (1) 1 (aggregate)2.8 3.2 2 2 (dimer″)″ 11.7 10.7 16.9 3 (monomer) 72.3 73.8 70.1 4 13.212.4 11.1 Lyo_phosphate (2) 1 (aggregate) 1.4 1.2 15.3 2 (dimer″)″ 10.18.9 32.7 3 (monomer) 74.7 78.4 43.9 4 13.9 11.6 8.1 Lyo_citrate (3) 1(aggregate) 0.9 0.6 1 2 (dimer″)″ 11.2 9.1 18.8 3 (monomer) 74.2 77.970.3 4 13.8 12.4 9.9

Consistent with the results seen in example 3, lyophilized rA13formulations displayed no increase in the amount of dimer oraggregate/denatured species after storage at 4° C. for at least 6months. Similarly, rA13 formulated and lyophilized with histidine orsodium citrate showed only minor levels of dimerization when stored at37° C. for at least 6 months. Furthermore, there was no indication thateither of these formulations resulted increased levels of aggregationand/or denaturation. Conversely, rA13 formulated and lyophilized withphosphate buffer displayed significant levels of dimerization andaggregation and/or denaturation, when stored at 37° C. for at least 6months.

E. Example 5 Biophysical Characterization of rA13 Formulations Stored at4° C. and 37° C.

rA13 samples formulated and aliquoted as in example 2 were stored ateither 4° C. or 37° C. for up to 6 months time. As a measure of theconformational stability of rA13 protein formulated as described above,stokes radii were determined by dynamic light scattering for thesolution (A) and lyophilized (B) rA13 formulations with histidine (FIG.6), phosphate buffer (FIG. 7), and sodium citrate (FIG. 8), at each ofthe indicated time points.

Furthermore, derived count rates for each of the liquid formulation,before storage, were determined by dynamic light scattering. As can beseen in FIG. 12, there is a discontinuity in the change of the stokesradii, and thus rA13 stability, above about 35° C. for the histidine andsodium citrate formulations, and above about 40° C. for the phosphateformulation. Furthermore, although the phosphate formulation has alarger stokes radius than the histidine or sodium citrate formulationsat temperatures below about 37° C., the phosphate formulation appears tobe slightly more stable at temperatures between about 40° C. and 50° C.

Lyophilized samples formulated with histidine were stored at 4° C. or37° C. for 3 months, and then reconstituted in sterile water. In orderto characterize whether or not local or global conformational changesoccurred in reconstituted rA13, fourier transform infrared spectroscopywas performed for the samples stored at 4° C. and 37° C. for 3 months,and compared to freshly formulated and lyophilized A13. As can be seenin FIG. 9, the absorbance spectrums for all three samples overlay nearlyperfectly, suggesting that no conformational transitions are occurringin rA13 stored as a lyophilized formulation in histidine at 4° C. or 37°C. for 3 months.

rA13 formulations stored at 4° C. or 37° C. for 6 months were alsocharacterized by reverse phase-high-performance liquid chromatography(rp-HPLC). rp-HPLC allows for the separation of closely related proteinmolecules and thus in many instances can differentiate between modifiedversions of the same polypeptide, e.g. charge-modified,partially-degraded, or partially-unfolded. As can be seen in FIG. 10,all rA13 formulations appear to be stable for at least 6 months at 4°C., consistent with several results seen above.

rA13 samples formulated as in example 2 were next analyzed bydifferential scanning calorimetry to further characterize the stabilityof the rA13 protein in the various formulations. The bimodal nature ofthe heat capacity curve generated in FIG. 11, suggests that two domainsof the protein may unfold separate of each other, with the firstunfolding transition occurring at a temperature between about 54° C. and55° C. The unfolding temperatures for rA13 are similar for the variousformulations. However, the increased enthalpy of transition, asindicated by the larger area beneath the endotherm, for the phosphateformulation, and to a lesser extent the citrate formulation, suggestslightly stabilized formulations of A13.

F. Example 6 Characterization of Dimer Formation in rA13 Formulations

In order to determine the effect of stabilizing agents on the oligomericstate of rA13 in solution, rA13 was formulated in 20 mM histidine (pH7.0), 150 mM NaCl, and 0.05% polysorbate 80 with different combinationsof sucrose (0% to 2%), mannitol (0% to 3%), and calcium (0 mM or 2 mM).The various formulations were then analyzed by gel filtration over aSepharose 6 GL column (GE Healthcare). As seen in FIG. 13, variouscombinations of the stabilizing agents were able to reduce the amount ofA13 dimer formation by about 50%. The percentages of aggregates,dimers/oligos, and monomers for each formulation is provided in Table14.

TABLE 14 Formulations used to determine the effect of various bufferingagents of the formulation of ADAMTS13. Man- Aggre- Dimer/ Mono- CaCl₂Sucrose nitol gate Oligomer mer Lot [mM] [g/l] [g/l] [%] [%] [%]rADAMTS008-1 — 10 20 0.854 46.34 53.04 rADAMTS008-2 2 10 20 1.664 42.0856.94 rADAMTS008-3 — 10 — 1.932 50.54 48.16 rADAMTS008-4 2 10 — 1.97941.57 57.14 rADAMTS008-5 — 10 10 1.820 49.8  49.13 rADAMTS008-6 2 10 101.748 40.98 58.01 rADAMTS008-7 — 10 30 1.645 47.07 51.88 rADAMTS008-8 210 30 2.242 41.23 57.33 rADAMTS008-9 — — — 1.540 51.62 47.39rADAMTS008-10 2 — — 1.429 39.49 59.62 rADAMTS008-11 — — 20 1.496 45.7 53.41 rADAMTS008-12 2 — 20 1.428 39.93 59.18 rADAMTS008-13 — 20 — 2.08348.13 50.56 rADAMTS008-14 2 20 — 1.997 41.43 57.34 rADAMTS008-15 — 20 201.904 46.77 52.06 rADAMTS008-16 2 20 20 1.866 41.66 57.2  rADAMTS008-17— 20 30 1.956 45.97 52.81 rADAMTS008-18 2 20 30 2.126 41.68 57.07

G. Example 7 Effect of pH on ADAMTS13 Formulation Stability

In order to determine the effect of pH on the stability of rA13 insolution, rA13 was dialyzed into buffer containing 20 mM histidine and150 mM NaCl, adjusted to a range of pH's between 5.5 and 9.5, and storedat either 4° C. or 40° C. for 24 hours. Stability was measured by rA13antigen concentration, as determined by ELISA, and rA13 activity byFRETS-VWF73 assay, at 24 hours. As seen in Table 3 and FIG. 14,rADAMTS13 is relatively stable for 24 hours at 4° C. when formulated ina solution having a pH between 5.5 and 9.5.

TABLE 3 ADAMTS13 stability in liquid formulation when stored at 4° C.for 24 hours. rADAMTS003 FRETS ADAMTS:Antigen 5012-02-0552 5016-01-0351[U/ml] [%] [U/ml] [%] Starting material 260792 100% 2208 100% pH 5.5216356 83% 1468 66% pH 6.5 399216 153% 2042 92% pH 7.5 358512 137% 217899% pH 8.5 176649 68% 2179 99% pH 9.5 110622 51% 1696 77%

TABLE 4 ADAMTS13 stability in liquid formulation when stored at 40° C.for 24 hours. rADAMTS002 FRETS ADAMTS:Antigen Prot: 5012-02-02975016-01-0297 [U/ml] [%] [U/ml] [%] Starting Material 281855 100% 1825100% pH 5.5 4264 2% 1225 67% pH 6.5 12111 4% 1836 101% pH 7.5 7190 3%2106 115% pH 8.5 4729 2% 1455 80% pH 9.5 <2500 1437 79%

H. Example 8 Effect of Low Levels of CaCl₂ on ADAMTS13 FormulationStability

In order to determine the effect of low levels of CaCl₂ on the stabilityof rA13 in solution, rA13 was formulated in buffer containing 150 mMNaCl, 2% sucrose, 0.05% polysorbate 80, and 20 mM histidine at pH 7.0with (12B and D) and without (12A and C) 2 mM CaCl₂. The liquidformulations were stored at either 25° C. or 37° C. for three weeks.Stability of the formulation was then assessed by determining therelative FRETS-VWF73 activity at 0, 1, 2, and 3 weeks. As can be seen inFIG. 15, the addition of 2 mM CaCl₂ stabilized liquid rADAMTS13formulations stored at both 25° C. and 37° C., to varying degrees.Notably, addition of low levels of CaCl₂ to rADAMTS13 liquidformulations stored at 25° C. conferred about 95% enzyme activitystability for three weeks, as compared to an 80% loss of activity seenin formulations without CaCl₂.

I. Example 9 FRETS-VWF73 Activity of Oligomeric Species of RecombinantADAMTS13

In order to determine the enzymatic activity of the different oligomericspecies of ADAMTS13 protein isolated during purification, rA13 wasloaded onto a Superose 6 GL gel filtration column equilibrated with abuffer containing 20 mM Na₂HPO₄ (pH 7.5) and 500 mM NaCl. ADAMTS13molecular species were then separated by size and shape. Fractionseluting from the column were pooled according to their apparentoligomeric state, e.g. aggregated A13 (Pool 1), dimeric A13 (Pool 2),and monomeric A13 (Pool 3) (FIG. 16A). Further confirmation of theoligomeric state of ADAMTS13 was obtained by determining the averagestokes radius of the protein in each pool by dynamic light scattering(FIG. 16B).

The enzymatic activity of each pool was then determined by FRETS-VWF73assay. As can be seen in FIG. 16, the volumetric activity of theaggregated A13 species was about 218 mU/ml, the activity of dimeric A13was about 1.435 U/ml, and the activity of the monomeric A13 was about97.905 U/ml. After standardizing the activities for the volume of thepools, greater than 99% of the total activity is found in the elutionpool corresponding to the monomeric protein. When the activities arethen standardized for the total amount of protein, it can be seen thatthe monomeric protein pool has a higher specific activity than both thedimeric protein pool and the aggregated protein pool.

J. Example 10 Effect of NaCl Concentration on the Lyophilization ofADAMTS13 Formulations

In order to determine the effect of NaCl concentration on the productionof lyocakes, recombinant ADAMTS13 was formulated in buffer containing 2%sucrose, 0.05% polysorbate 80, and 20 mM histidine (pH 7.0) with varyingamounts of NaCl (0 to 150 mM). The ADAMTS13 formulations were thenlyophilized under standard conditions and visually inspected for thequality of the resulting lyocakes (FIG. 17). Lyocakes produced fromADAMTS13 formulations containing lower salt concentrations (0 to 60 mM;FIGS. 17A-C) had compact structures with smooth surfaces, while lyocakesproduced from high salt formulations (120 mM and 150 mM NaCl; FIGS. 17 Eand F) were porous and not compact, with a string-like or crackedappearance. ADAMTS13 formulations containing intermediate levels of NaCl(90 mM; FIG. 17D) were partially compact with a semi-porous orcrater-like surface.

A porous, non-compact lyocake indicates melting during thelyophilization process. Generally, high salt concentrations are used todecrease the collapse temperature (or glass temperature) that can resultin the partial melting of the frozen material during the primary dryingprocess. This can have a negative impact on protein aggregation levelsand/or the recovery of enzymatic activity. Therefore, a good lyocakeappearance usually correlates with better recovery of activity and lessaggregation of the respective formulated protein. Advantageously, thepresent invention provides ADAMTS13 formulations which allow for theproduction of high quality lyocakes. In certain embodiments, theinventive formulations provided herein allow for low salt formulationsof ADAMTS13, that are particularly stable during lyophilization,resulting in the formation of high quality lyocakes.

One concern with the use of low salt protein formulations is thatincreased protein aggregation may occur. To determine if this was thecase for the low salt ADAMTS13 formulations, the lyocakes produced abovewere reconstituted in de-ionized water and the aggregation states of thereconstituted ADAMTS13 proteins were analyzed by size exclusionchromatography. As can be seen in FIG. 18, the salt concentration of thelyophilized formulations had no effect on the aggregation state ofreconstituted ADAMTS13 protein (compare 0 mM NaCl with 150 mM NaCl).

To further validate the use of low salt ADAMTS13 formulations, theFRETS-VWF73 activity of various formulations ranging from 0 to 150 mMNaCl was determined. As can be seen in FIG. 19, the salt concentration,as well as the sucrose concentration, of the ADAMTS13 formulations didnot influence the activity of the recombinant ADAMTS13 protein. Theseresults suggest that low salt (i.e. 0 to 100 mM NaCl) formulations ofliquid and lyophilized recombinant ADAMTS13 proteins are well suited forpharmaceutical use.

K. Example 11 Lyophilized Formulation of ADAMTS13 in High Salt and LowSalt Buffers

To compare lyophilized formulations of recombinant ADAMTS13, high salt(150 mM NaCl) and low salt (30 mM NaCl) formulations were prepared. Boththe high salt and low salt formulations contained 20 mM histidine (pH7.0), 150 mM NaCl, 0.05% polysorbate 80, and 2 mM CaCl₂. The high saltformulation further contained 2% sucrose and 150 mM NaCl, while the lowsalt formulation further contained 1% sucrose, 3% mannitol, and 30 mMNaCl. The two formulations were then lyophilized under standardconditions. The resulting lyocake produced from the high salt ADAMTS13formulation was not compact, non-uniform, clumpy, and porous (FIG. 20A),while the lyocake produced from the low salt ADAMTS13 formulation wascompact, fairly uniform, and had a smooth surface (FIG. 20B). Theseresults suggest that low salt formulations of lyophilized ADAMTS13 arewell suited for pharmaceutical use.

L. Example 12 Purification of rADAMTS13 for Lyophilized Formulations

Recombinant ADAMTS13 was expressed in recombinant CHO cells and purifiedfrom the resulting conditioned media by POROS HS cation exchangechromatography. Collected fractions of rADAMTS13 eluted off of the HScolumn were then analyzed, by size exclusion chromatography using asuperose 6 GL: TC10/30 column, for dimerization and aggregation prior toand after gel filtration, by size exclusion chromatography (FIGS. 21Aand B, respectively) and dynamic light scattering (FIGS. 22A and B,respectively). As can be seen in FIG. 21A, nearly 20% of the totalrADAMTS13 in the HS-eluted fractions, prior to gel filtration, were inundesirable dimeric, monomeric, or aggregated states. In contrast,almost all of the rADAMTS13 pooled from the gel filtration step ismonomeric (FIG. 21B). Similarly, the average hydrodynamic radius of theHS-eluted rADAMS13 (about 20.5 nm; FIG. 22A) was nearly twice that ofthe rADAMTS recovered after size exclusion chromatography (about 12-13nm; FIG. 22B)

M. Example 13 Comparison of Standard and Extended Protocols forLyophilization of rADAMTS13 Formulations

In order to compare lyophilization of rADAMTS13 formulations containingvariable low salt (30 mM and 60 mM NaCl), calcium (2 mM and 4 mM CaCl₂),and sugar (1% sucrose and 1% trehalose) concentrations, rADAMTS13 waspurified by cation exchange chromatography and size exclusionchromatography, as above, and formulated in 20 mM histidine, 3%mannitol, 0.05% polysorbate 80, with all eight combinations of salt,calcium, and sugar given above. All vials had the same concentration ofADAMTS13 (190 μg/mL antigen). rA13 formulations 1 to 8 (Tables 5 and 6)were then lyophilized using either a standard (3 days; FIG. 23A) orextended (11 days; FIG. 23B) lyophilization protocol. Afterlyophilization, the lyocakes were visually inspected for desirablequalities (FIG. 24).

TABLE 5 Lyophilized rADAMTS13 formulations used in standardlyophilization protocol. rA13 Formulation NaCl Histidine CaCl₂ MannitolSucrose Trehalose Tween 80 (A LYO) (mM) (mM) (mM) (%) (%) (%) (%)Optical Evaluation 1 30 mM 20 mM 2 mM 3% 1% — 0.050% Beautiful, compactlyocake; smooth surface detached from wall 2 30 mM 20 mM 2 mM 3% — 1%0.050% Same as variant 1 3 30 mM 20 mM 4 mM 3% 1% — 0.050% Same asvariant 1 4 30 mM 20 mM 4 mM 3% — 1% 0.050% Same as variant 1 5 60 mM 20mM 2 mM 3% 1% — 0.050% Beautiful, compact lyocake; smooth surface,periphery porous and easily detached from wall 6 60 mM 20 mM 2 mM 3% —1% 0.050% Same as variant 5; surface easily detached from lyocake 7 60mM 20 mM 4 mM 3% 1% — 0.050% Same as variant 5; peripherypulverulent/flocculent 8 60 mM 20 mM 4 mM 3% — 1% 0.050% Same as variant5; surface detached from lyocake; periphery pulverulent

TABLE 6 Lyophilized rADAMTS13 formulations used in extendedlyophilization protocol. rA13 Tween Formulation NaCl Histidine CaCl₂Mannitol Sucrose Trehalose 80 (B LYO) (mM) (mM) (mM) (%) (%) (%) (%)Optical Evaluation 1 30 mM 20 mM 2 mM 3% 1% — 0.050% Beautiful, compactlyocake; smooth surface broken from periphery 2 30 mM 20 mM 2 mM 3% — 1%0.050% Same as variant 1 3 30 mM 20 mM 4 mM 3% 1% — 0.050% Same asvariant 1 4 30 mM 20 mM 4 mM 3% — 1% 0.050% Same as variant 1 5 60 mM 20mM 2 mM 3% 1% — 0.050% Same as variant 1 6 60 mM 20 mM 2 mM 3% — 1%0.050% Same as variant 1 7 60 mM 20 mM 4 mM 3% 1% — 0.050% Beautiful,compact lyocake; porous surface broken from wall; periphery flocculent 860 mM 20 mM 4 mM 3% — 1% 0.050% Same as variant 1; surface broken

To determine the multimeric state of ADAMTS13 in each of theformulations, lyophilized protein was reconstituted in deionized waterand analyzed by dynamic light scattering. As can be seen in FIG. 25,lyophilized formulations prepared by using both standard and extendedlyophilization protocols, yielded primarily monomeric ADAMTS13 molecules(peak at about 12-13 nm) after reconstitution. Although the ADAMTS13peak corresponding to aggregated protein (between 100 and 110 nm)appears to be about half as high as the monomer peak, one of ordinaryskill will recognize that the y-axis is measuring intensity, rather thantotal mass, and as such the graph greatly exaggerates the percentage ofADAMTS13 species that are aggregated. It is also seen that formulationsprepared with extended lyophilization protocols (dashed lines) containless aggregated ADAMTS13 than formulations prepared with standardlyophilization protocols (unbroken lines), after reconstitution indeionized water.

In order evaluate the stability of the lyophilized rADAMTS13 in each ofthe formulations presented above, the lyocakes produced by the standardand extended protocols were used to prepare a series of vials forstorage under various temperatures for up to 36 months. As outlined inTable 7, vials containing either 5 mg or 10 mg lyophilized rADAMTS13were prepared for storage at +2-+8° C. (i.e. under standardrefrigeration conditions), 30° C. (room temperature), and 40° C. Allvials were tested by FTIR, SE-HPLC and DLS. Also FRETS activity andADAMTS13 antigen were tested for all samples. Lyophilized ADAMTS13formulations can then be tested for FRETS-VWF73 activity and antigenstability, as outlined above, at the indicated time points to determinethe stability of the protein in the various lyophilized formulations.Tables 8 and 9 provide the results of FRETS-VWF73 activity assays ofreconstituted ADAMTS13 formulations generated by the standard andextended lyophilization protocols, respectively. Similarly, antigenrecovery is given in tables 10 and 11, for samples generated with thestandard and extended lyophilization protocols, respectively.

TABLE 7 Experimental set-up for testing the stability of lyophilizedADAMTS13 formulations. Set-up corresponds to both A and B typelyophilizations. Vial buffer 0 M 1 M 2 M 3 M 4 M 6 M 9 M 12 M 15 M 18 M24 M 30 M 36 M -1B −80° C. 30 +2-+8° C. 20 10 5 5 5 5 5 5 5 5 5 RT 5 105 5 5 5 40° C. 5 5 10 5 5 -2B −80° C. 30 +2-+8° C. 20 10 5 5 5 5 5 5 5 55 RT 5 10 5 5 5 5 40° C. 5 5 10 5 5 -3B −80° C. 30 +2-+8° C. 20 10 5 5 55 5 5 5 5 5 RT 5 10 5 5 5 5 40° C. 5 5 10 5 5 -4B −80° C. 30 +2-+8° C.20 10 5 5 5 5 5 5 5 5 5 RT 5 10 5 5 5 5 40° C. 5 5 10 5 5 -5B −80° C. 30+2-+8° C. 20 10 5 5 5 5 5 5 5 5 5 RT 5 10 5 5 5 5 40° C. 5 5 10 5 5 -6B−80° C. 30 +2-+8° C. 20 10 5 5 5 5 5 5 5 5 5 RT 5 10 5 5 5 5 40° C. 5 510 5 5 -7B −80° C. 30 +2-+8° C. 20 10 5 5 5 5 5 5 5 5 5 RT 5 10 5 5 5 540° C. 5 5 10 5 5 -8B −80° C. 30 +2-+8° C. 20 10 5 5 5 5 5 5 5 5 5 RT 510 5 5 5 5 40° C. 5 5 10 5 5

TABLE 8 FRETS-VWF73 activity in lyophilized ADAMTS13 formulationsprepared by standard lyophilization protocols. [U/ml] [%] [U/ml] [%] 0 M1 M [U/ml] [%] [U/ml] [%] [U/ml] [%] [U/ml] [%] [U/ml] [%] WH MW WH 2 M3 M 4 M 6 M 9 M -1 A  4° C. 126 88 107 100% — — — — 107.90  86% 106.00 84% 108.40  85% 104.90  83% 30° C. — — 105  83% 100.70  80% 95.30  76%95.70  75% 99.10  79% 40° C. 98  92% 127 100% 81.50  65% 86.70  69%88.40  70% — — -2 A  4° C. 101 100 101 100% — — — — 99.4  98% 99.7  98%98.2  97% 101.2 100% 30° C. — — 98  97% 100.1  99% 93  92% 92.1  91%99.3  98% 40° C. 99  98% 95  94% 93.3  92% 93.5  92% 83.3  82% — — -3 A 4° C. 117 119 118 100% — — — — 101.9  87% 99.1  84% 104.4  89% 105.8 90% 30° C. — — 105  90% 97.9  83% 94.1  80% 93.5  80% 98.5  84% 40° C.114  96% 101  86% 91.7  78% 87.2  74% 87  74% — — -4 A  4° C. 113 100106 100% — — — — 97.7  87% 99.9  89% 103  91% 101.8  90% 30° C. — — 107 95% 97.9  87% 91.9  82% 90  80% 96.5  86% 40° C. 100  94% 122 108% 95.3 85% 89.3  79% 91.2  81% — — -5 A  4° C. 109 97 103 100% — — — — 94.1 86% 100.2  92% 107.7  99% 104.5  96% 30° C. — — 125 115% 93.9  86% 91.6 84% 89.2  82% 98  90% 40° C. 106 103% 107  98% 84.5  77% 74.1  68% 80.1 73% — — -6 A  4° C. 93 81 87 100% — — — — 97.2 104% 94.7 101% 104.5112% 102.7 110% 30° C. — — 103 111% 94.4 101% 91.5  98% 91.6  98% 97.3104% 40° C. 85  97% 90  96% 89.8  98% 84.7  91% 82  86% — — -7 A  4° C.111 102 106 100% — — — — 98.9  89% 100.2  90% 103  93% 100.1  90% 30° C.— — 105  94% 90.6  82% 89.4  81% 85.2  77% 94.4  85% 40° C. 105  99% 114103% 85.1  77% 77.8  70% 79.6  72% — — -8 A  4° C. 101 126 113 100% — —— — 104 103% 100.3  99% 101.6 101% 101.5 101% 30° C. — — 126 125% 89.6 89% 92.9  92% 94.2  93% 92.8  92% 40° C. 98  86% 99  98% 88.1  87% 82.7 82% 89.3  89% — —

TABLE 9 FRETS-VWF73 activity in lyophilized ADAMTS13 formulationsprepared by extended lyophilization protocols. [U/ml] [%] [U/ml] [%][U/ml] [%] [U/ml] [%] [U/ml] [%] [U/ml] [%] [U/ml] [%] 0 M 1 M 2 M 3 M 4M 6 M 9 M -1 B  4° C. 112 100% — — — — 101.70 91% 102.40 92% 105.60 95%100.70 90% 30° C. — — 97 87% 93.30 84% 95.60 86% 102.30 92% 93.00 83%40° C. 108 97% 97 87% 101.40 91% 96.80 87% 86.10 77% — — -2 B  4° C. 104100% — — — — 93.9 91% 99.1 96% 107.3 104%  98.8 95% 30° C. — — 86 83%93.4 90% 99.2 96% 101.1 98% 93.9 91% 40° C. 106 102%  87 84% 104.2 101% 94.2 91% 92.5 89% — — -3 B  4° C. 111 100% — — — — 91.7 83% 101.2 92%105.1 95% 103.8 94% 30° C. — — 94 85% 94.2 85% 95 86% 102.2 92% 91.7 83%40° C. 98 88% 108 98% 102.6 93% 92.7 84% 94.6 86% — — -4 B  4° C. 135100% — — — — 91.8 68% 97.6 72% 102.9 76% 100.5 74% 30° C. — — 104 77%97.8 72% 97.8 72% 102.8 76% 95.2 70% 40° C. 87 64% 100 74% 98.6 73% 92.668% 83.8 62% — — -5 B  4° C. 111 100% — — — — 86.2 78% 97.8 88% 107 97%95.5 86% 30° C. — — 97 87% 92.6 84% 93.1 84% 101 91% 89.1 80% 40° C. 8577% 82 74% 98.2 89% 82.6 75% 78 70% — — -6 B  4° C. 116 100% — — — —88.8 77% 99.2 86% 105.2 91% 101.5 88% 30° C. — — 90 78% 97.5 84% 100.287% 102.9 89% 91.4 79% 40° C. 95 83% 93 80% 100.8 87% 85.8 74% 81.4 70%— — -7 B  4° C. 136 100% — — — — 85.8 63% 96.6 71% 102.6 76% 98 72% 30°C. — — 97 71% 94.1 69% 95.7 71% 97.9 72% 88.8 66% 40° C. 90 66% 93 69%98.8 73% 84.4 62% 79.8 59% — — -8 B  4° C. 119 100% — — — — 88.3 74%93.1 78% 100.1 84% 98.8 83% 30° C. — — 108 91% 95.7 80% 95.4 80% 101 85%93.7 79% 40° C. 106 89% 88 74% 102.5 86% 89.4 75% 80.5 67% — —

TABLE 10 Recovery of A13 antigen in lyophilized ADAMTS13 formulationsprepared by standard lyophilization protocols. [μg/ml] [%] [μg/ml] [%][μg/ml] [%] [μg/ml] [%] [μg/ml] [%] [μg/ml] [%] [μg/ml] [%] 0 M 1 M 2 M3 M 4 M 6 M 9 M -1 A  4° C. 144 100% — — — — 147 103% 148 103% 122 85%129 90% 30° C. — — 131  91% 139  97% 132  92% 136 95% 136 95% 40° C. 151105% 140  97% 137  95% 125  87% 107 75% — — -2 A  4° C. 155 100% — — — —145  93% 137  88% 125 81% 137 88% 30° C. — — 142  91% 137  88% 133  86%154 99% 133 88% 40° C. 157 101% 141  91% 137  88% 123  79%  97 63% — —-3 A  4° C. 143 100% — — — — 149 104% 146 102% 121 85% 136 95% 30° C. —— 141  99% 132  92% 134  94% 132 93% 136 95% 40° C. 164 115% 134  94%130  91% 139  97% 109 76% — — -4 A  4° C. 155 100% — — — — 152  98% 142 91% 130 84% 126 81% 30° C. — — 142  92% 133  85% 125  80% 129 83% 14392% 40° C. 157 101% 136  88% 131  84% 127  82% 113 73% — — -5 A  4° C.137 100% — — — — 140 102% 154 113% 118 86% 125 91% 30° C. — — 143 105%121  88% 126  92% 141 103%  132 97% 40° C. 148 108% 139 102% 122  89%157 115% 112 82% — — -6 A  4° C. 138 100% — — — — 150 109% 134  97% 12490% 122 88% 30° C. — — 145 105% 137  99% 125  90% 143 103%  139 101% 40° C. 147 107% 142 103% 127  92% 119  86% 124 90% — — -7 A  4° C. 145100% — — — — 148 102% 153 105% 137 95% 126 87% 30° C. — — 137  95% 130 90% 125  86% 148 102%  140 97% 40° C. 150 104% 142  98% 128  89% 137 95% 119 82% — — -8 A  4° C. 146 100% — — — — 135  92% 146 100% 138 94%136 93% 30° C. — — 131  89% 137  94% 122  84% 170 117%  139 95% 40° C.152 104% 142  97% 123  84% 129  88% 122 83% — —

TABLE 11 Recovery of A13 antigen in lyophilized ADAMTS13 formulationsprepared by extended lyophilization protocols. [μg/ml] [%] [μg/ml] [%][μg/ml] [%] [μg/ml] [%] [μg/ml] [%] [μg/ml] [%] [μg/ml] [%] 0 M 1 M 2 M3 M 4 M 6 M 9 M -1 B  4° C. 112 100% — — — — 133 118% 137 122% 129 115%158 141% 30° C. — — 144 129% 128 115% 128 114% 145 130%  0% 40° C. 138123% 136 121% 131 117% 132 118% 128 115% — — -2 B  4° C. 120 100% — — —— 132 111% 136 114% 131 109% 164 137% 30° C. — — 137 115% 128 107% 124103% 142 119%  0% 40° C. 130 109% 143 120% 147 123% 137 115% 134 112% —— -3 B  4° C. 134 100% — — — — 132  99% 138 103% 133  99% 170 126% 30°C. — — 140 105% 120  90% 123  92% 142 105%  0% 40° C. 128  95% 137 102%132  98% 138 103% 128  95% — — -4 B  4° C. 138 100% — — — — 143 103% 141102% 134  97% 164 118% 30° C. — — 153 111% 142 103% 126  91% 156 112% 0% 40° C. 123  89% 135  98% 137  99% 142 103% 138  99% — — -5 B  4° C.109 100% — — — — 139 128% 135 124% 132 121% 171 157% 30° C. — — 137 125%121 111% 126 116% 140 128%  0% 40° C. 122 112% 132 121% 135 123% 129118% 125 114% — — -6 B  4° C. 112 100% — — — — 146 130% 140 126% 127113% 174 156% 30° C. — — 140 126% 124 111% 126 113% 142 127%  0% 40° C.129 115% 144 129% 135 120% 142 127% 131 117% — — -7 B  4° C. 123 100% —— — — 144 117% 146 118% 131 106% 171 138% 30° C. — — 149 121% 127 103%129 105% 136 110%  0% 40° C. 128 103% 135 110% 143 116% 136 110% 130106% — — -8 B  4° C. 115 100% — — — — 120 104% 130 113% 131 114% 172149% 30° C. — — 132 114% 126 109% 127 110% 137 119%  0% 40° C. 124 107%141 123% 136 118% 134 118% 128 111% — —

Tables 17 and 18 summarizes the FTIR results of the different lyoprograms for rADAMTS022-1 formulations. Neither time nor temperature hada major influence on secondary structures. Also the lyo program did notchange structural elements, apart from the apparent decrease of alphahelix at 40° C. for the standard program A which was not seen for thelonger lyo program B.

TABLE 17 Secondary structure of lyophilized rADAMTS13 formulation 22 -1Aevaluated by FTIR. Prediction α-helix [%] Prediction β-sheet [%]rADAMTS022-1A 4° C. 30° C. 40° C. 4° C. 30° C. 40° C. start 6.7 37.7 1month 11.7 36.6 2 months 11.7 11.3 37.9 36.4 3 months 10.9 10.7  8.936.8 38.4 35.7 4 months 14.6 12.2  7.4 37.9 39.1 36.1

TABLE 18 Secondary structure of lyophilized rADAMTS13 formulation 22 -1Bevaluated by FTIR. Prediction α-helix [%] Prediction β-sheet [%]rADAMTS022-1B 4° C. 30° C. 40° C. 4° C. 30° C. 40° C. start 10.8 36.6 1month 11.1 38.6 2 months  9.3  8.9 37.8 38.4 3 months 11.2 13.7 12.638.9 38.4 34.3 4 months  6.6 12.8 10.5 35.9 38.5 37.1

The oligomeric state of ADAMTS13 in the 1A and 1B lyophilizedformulations was analyzed by dynamic light scattering over the course of6 months of storage at 4° C., 30° C., and 40° C. As seen in FIGS. 37 and38, higher aggregate levels were observed in the samples prepared withthe extended lyophilization program. However, aggregation levels did notsubstantially increase over the storage time.

Similarly, size exclusion HPLC did not show no major changes between thedifferent lyo programs for rADAMTS022 formulations after 6 months (datanot shown). The analysis show that no aggregates were induced afterstorage at 4° C., 30° C., and 40° C. over the course of 3 months.

N. Example 14 Expression and Purification of Recombinant Human ADAMTS13

Recombinant ADAMTS-13 is generated by a recombinant Chinese HamsterOvary (CHO) cell clone in a fermentation process in suspension culture.The growth medium, developed by Baxter and is both free of human oranimal derived substances and recombinant proteins. Examples of thesetypes of growth mediums useful for the expression of ADAMTS13 can befound, for example, in U.S. patent application Ser. No. 12/847,999. Themanufacturing process utilizes a continuous (chemostat) cell culturemethod. The purification process starts with an initial cell removalstep by filtration. The cell free product of up to 4 subsequent days iscombined to produce one downstream batch. The pooled, filtered harvestsare concentrated by an ultra/diafiltration and then subjected to asolvent detergent virus inactivation step. Further purification includesa chromatographic capture step (Anion Exchange), a nanofiltration step(second virus reduction step), a negative chromatography step(hydroxyapatite) followed by a mixed mode chromatography (Capto MMC) anda final chromatographic concentration and pre-formulation step (CationExchange). The pre-formulated bulk drug substance (BDS) is frozen at−60° C. in a temperature-controlled freezer.

O. Example 15 FRETS-VWF73 Assay for ADAMTS13 Activity

The proteolytic activity of ADAMTS13 was measured against afluorescence-quenching substrate (FRETS-VWF73, Peptides Institute, Inc;Osaka, Japan) according to the assay description of the manufacturer.Briefly, rADAMTS13 samples were diluted (in 100 μL total volume) inbuffer containing 5 mM Bis-Tris, 25 mM CaCl2, and 0.005% Tween20 andtransferred into a black microtiter plate. Samples were measured againsta reference curve of diluted human plasma samples (from 80 to 5 mU/mLplasma). The reaction was started by adding the substrate (100 μL,FRETS-VWF73; 2 μM final concentration) and fluorescence was measuredevery two minutes for 45 minutes in a fluorescence spectrophotometerwith lex=360 nm and lem=460 at 30° C. (FLx800, Bio Tek). The activityresults were read off a reference curve of human plasma. Data areexpressed as Unit/mL. Normal human plasma was regarded as 1 Unit/mL.

P. Example 16 ADAMTS13 Antigen ELISA

ADAMTS13 containing samples are analyzed in an ELISA assay usingpolyclonal rabbit IgG directed against human ADAMTS13 both as captureand in its labeled form as detection antibody. ADAMTS13 antigen presentin the sample is captured by the ADAMTS13-specific antibody coated on a96-well microtiter plate. Samples were diluted in buffer (250 mM Tris,350 mM NaCl, 0.5% BSA, 0.1% Tween 20) and transferred to the coatedmicrotiter wells. Bound ADAMTS13 antigen is detected with HRP-conjugatedrabbit anti-ADAMTS13 IgG using TMB as substrate (Thermo Art: #34021).Absorbance was measured in a spectrophotometer (TECAN SpectraFluorPlus,Tecan Sales Austria, Gröding, Austria) at 1=450 nm. ADAMTS13 antigenconcentrations (expressed in μg/mL) were calculated from a referencestandard using dilutions ranging from 250 to 7.81 ng/mL of recombinantADAMTS13 that was purified from stably transfected HEK293 cell cultureharvests.

Q. Example 17 Size Exclusion High Performance Liquid Chromatography(SE-HPLC) Analysis of ADAMTS13

SE-HPLC was performed using an AKTA Purifier “900-series” (GEHealthcare). The system was equipped with a Superose 12 GL column (GEHealthcare, TC10/30) which was run at a constant flow rate of 0.3 mL perminute at room temperature. As running buffer 20 mM Tris, 100 mM sodiumacetate, 500 mM sodium chloride, pH 7.4 was used. The sample wascentrifuged (Centrifuge 5415C, Eppendorf, Vienna, Austria) for 5 min at10,000 rpm and 100 μL were applied automatically by an autosampler. Theabsorbance of the column effluent was measured continuously at 280 nm.

R. Example 18 Dynamic Light Scattering (DLS) Analysis of ADAMTS13

Dynamic light scattering (DLS) was performed using a Malvern NanoZetasizer ZS (Malvern Instruments Ltd Enigma Business Park, GrovewoodRoad, Malvern, Worcestershire, UK. WR14 1XZ) and a Haake Rheostress 1(Thermo Fisher Scientific, Karlsruhe, Germany) equipped with a cone with60 mm diameter/0.5° angle for buffer viscosity measurements.

All samples were centrifuged (Centrifuge 5415C, Eppendorf, Vienna,Austria) for 5 min at 10,000 rpm to determine the hydrodynamic diameterof a protein. 60 μL of sample were filled into a ZEN0040 disposablemicro cuvette and viscosity of buffer was determined by Rheostress 1.This parameter is used for analyzing effective size of proteins by DLS.Operation temperature was 25° C. with an equilibration time of 2minutes. The proteins angle was set to 173° backscatter to measure thesize of and 3 runs per sample were performed to average the results.

Samples were measured by increasing temperature mode to monitor theinfluence of temperature on a protein. Measurement procedure was similarto a normal size measurement, except for different temperatures with anincreasing value of 1° C./min from 15° C. to 80° C. and an equilibrationtime of 2 min. A DTS2145 low volume glass cuvette was used for thesetemperature ramps.

S. Example 19 Fourier-Transformed Infrared Spectroscopy (FTIR) Analysisof ADAMTS13

Fourier-transformed infrared spectroscopy was performed using the FTIRspectroscope TENSOR 27 (Bruker Optik GmbH, 76275 Ettlingen, Germany)equipped with a BioATR II cell working in attenuated total reflectionmode. This instrument configuration can be used to analyze theconformation of protein formulations.

During an operating temperature of 20° C., 20 μL of water were filledinto the cell and were measured for background scan. Afterwards 20 μLbuffer were filled into the cell and measured against background scan,to subtract water from buffer. This procedure was repeated with bufferas background scan for measuring the sample (measurement range offrequency: 4000-650 cm⁻¹). An interferogram was generated and translatedinto a transmission spectrum. Different spectra were corrected for sameoffset and were normalized to the same protein concentration.Additionally secondary structure (% α-helix, % β-sheet) of the proteinwas calculated by the evaluation software (OPUS 6.0/Bruker) whichcontained a database of ˜40 proteins of known secondary structure.

T. Example 20 Photoirradiation Analysis of ADAMTS13 Stability

Photostability testing was performed using the Atlas Suntest CPS+Photostability Chamber (Chicago, Ill., USA), according to SOPVN-09-45058 TB. The CPS+ monitors and controls irradiance, blackstandard temperature and chamber air temperature.

Photostability was tested in the same formulation as was used for shearand freeze-thaw experiments (Table 12). The samples and controls weretested by DLS and SE-HPLC. The results are shown in FIG. 31 and FIG. 32.Both formulations (liquid and Lyo) showed the same results by SE-HPLC.The monomer ADAMTS13 peak decreased and the dimer peak increased withirradiation time. Hydrodynamic diameter of liquid and lyophilizedformulation also slightly increased with time (FIG. 32). The lyophilizedformulation irradiated for 10 h (as recommended by ICH Q1B) did not showany difference to the light-protected control. FRETS activity monitoredduring photo-irradiation rapidly decreased in liquid but less inlyophilized formulations as shown in FIG. 33. Even after four-foldphotoirradiation dose, as recommended by ICH guideline Q1B whichcorresponds to 4.8M lux hours, more than 50% of FRETS activity remained.

The “control” vials were wrapped in aluminium foil to eliminate exposureto light and placed in the photostability chamber along with the testsamples. All samples were placed horizontally. An irradiation time of 10h corresponded to 1.2 to 1.8 million lux h and 765 W/m² UV light.Samples were removed from the photostability chamber after 10 h and thespectrum (190-800 nm) and pH value were measured.

U. Example 21 Mechanical Stress Analysis of ADAMTS13 Stability

Shear stress was applied with a Haake Rheostress1 (Thermo ElectronKarlsruhe GmbH, Karlsruhe, Germany) using a cone with 60 mm diameter anda 0.5° angle. Temperature was set to 25.0° C.

Briefly, recombinant human ADAMTS13, prepared as described above, wasformulated as in Table 12. 500 μL of ADAMTS13 sample per run wereapplied and stressed at 50, 100, 150, 200, 250, 300, 350, 500 and 600rpm for 15 min. Then the sample was transferred to an Eppendorf vial.DLS measurements were applied to monitor potential shear stress inducedpartial unfolding or aggregation. As shown in FIG. 28, aggregates wereformed at a shear stress of 200 rpm.

TABLE 12 Formulation used for mechanical stress analysis of ADAMTS13stability. Buffer substances BAX930 FL NaCl [mM] 150 Histidine [mM] 20Saccharose [%] 2 Polysorbate 80 [%] 0.05 pH 7.0

V. Example 22 Effects of Various Buffering Agents on ADAMT13Formulations

To determine the effect various buffers had on the stability andconformation of ADAMTS13 formulations, recombinant human ADAMTS13,expressed and purified as described above, was dialyzed againstdifferent buffers (Table 13) and analyzed by Dynamic light scattering(DLS) to identify basic buffer preferences of ADAMTS13. As can be seenin FIG. 27, the smallest hydrodynamic diameters of ADAMTS13 were foundwhen the protein was formulated in either histidine or HEPES buffers.

TABLE 13 Formulations used to determine the effect of various bufferingagents of the formulation of ADAMTS13. Buffer substances -1 -2 -3 -4 -5NaCl [mM] 150 150 150 150 150 Histidine [mM] 20 — — — — Hepes [mM] — 20— — — Sodium phosphate [mM] — — 20 — — Citrate [mM] — — — 20 — EDTA [mM]— — — — 20 pH 7.5 7.5 7.5 7.5 7.5 *All formulations contained 2% sucroseand 0.05% polysorbate 80.

W. Example 23 Freeze-Thaw Analysis of ADAMTS13 Stability

The same formulation (Table 12) as used for shear stress experiments wasagain used to investigate the behavior of the protein during freeze-thawstress. Two freeze-thaw conditions −20° C./RT and −80° C./+37° C. werechosen. FIG. 29 summarizes the results of the FRETS activitymeasurements. All samples are stable within the range of the assay'svariation (25% relative standard deviation). No FRETS activity loss wasseen even after a 5-fold freeze-thaw cycle. The stability of BAX930 inthis liquid formulation is confirmed by the results of the DLS andSE-HPLC measurements. No increase in the intensity of the aggregationpeaks (between 100 and 1000 nm) by DLS was observed independent offreeze-thaw conditions (FIG. 30). Similarly, SE-HPLC did not show anincrease of the aggregation level after repeated freeze thaw cycles at−80° C./+37° C. but some increase at −20° C./RT (data not shown).

X. Example 24 Effect of Calcium and Zinc on the FRETS Activity ofADAMTS13

Recombinant human ADAMTS13, prepared as described above, was treatedwith 10 mM EDTA and then dialyzed against buffer containing 20 mMhistidine and 190 mM NaCl (pH 7.5). After dialysis, CaCl₂ and ZnCl₂ wereadded back to the formulations at different concentrations. The activityof ADAMTS13 formulations containing different concentrations of calciumand zinc were then tested for FRET activity as described above. As canbe seen in FIG. 34, increasing levels of calcium increased the recoveryof FRETS activity. Near maximum activity was achieved with the inclusionof 4 mM CaCl₂, both in the presence and absence of zinc. At intermediateconcentrations of calcium (2 mM and 4 mM), the addition of zinc provideda modest increase in FRETS activity.

Y. Example 25 Influence of Salt and Sugar Concentration on Aggregationin Lyophilized ADAMTS13 Formulations

To further investigate the effect that salt and sugar concentrationshave on lyophilized formulations of ADAMTS13, the oligomeric state ofseveral lyophilized formulations was determined after reconstitutionwith deionized water. Briefly, recombinant human ADAMTS13 was producedas described above. Protein samples were then formulated with 20 mMhistidine (pH 7.0), 2 mM calcium chloride, and 0.05% polysorbate 80 withthe sodium chloride and sucrose levels given in Table 15. Theformulations were then lyophilized, as described above, andreconstituted with deionized water. Oligomeric characteristics were thendetermined by SE-HPLC analysis, the results of which are shown in Table15 and FIG. 35. As can been seen in formulations containing low sugarconcentrations, high salt levels (150 mM) increase ADAMTS13 aggregationand reduce the monomeric content of the formulation.

TABLE 15 Influence of salt and sugar concentration on aggregation inlyophilized ADAMTS13 formulations measured by SE-HPLC peak area. Osmo-Aggre- Dimer/ Mono- NaCl Sucrose larity gate Oligomer mer Lot [mM] [g/l][g/l] [%] [%] [%] rADAMTS016-1 — 20  80.5 —  8.3 91.9 rADAMTS016-2  3020 141.0 — 10.8 89.2 rADAMTS016-3  60 20 196.5 —  9.4 90.6 rADAMTS016-4 90 20 258.0 —  9.2 90.8 rADARITS016-5 120 20 307.5 —  9.9 90.1rADAMTS016-6 150 20 359.5 —  9.5 90.5 rADAMTS016-7  60 15 175.5 — 10.090.0 rADAMTS016-8 150 15 341.0 —  9.6 90.4 rADAMTS016-9  60 10 163.5 — 9.1 90.9 rADAMTS016-10 150 10 328.5 1.1  9.8 89.0 rADAMTS016-11  60  5147.0 — 10.0 90.0 rADAMTS016-12 150  5 314.5 0.7 10.4 88.8

The same formulation shown in Table 15 were then used to evaluate thequality of lyocake produced after lyophilization of ADAMTS13formulations. As summarized in Table 16, and shown in FIG. 36, thepresence of high concentrations of sodium chloride (150 mM) resulted inno lyocake. Conversely, the best lyocakes were obtained withformulations containing between 0 mM and 60 mM sodium chloride in thepresence of 2% sucrose.

TABLE 16 Quality of lyocake produced for various formulations ofrecombinant human ADAMTS13. Lot Lyocake quantification rADAMTS016-1compact lyocake, smooth surface and detached off from glass wallrADAMTS016-2 compact lyocake, smooth surface and detached off from glasswall rADAMTS016-3 compact lyocake, smooth surface and detached off fromglass wall rADAMTS016-4 compact, porous lyocake, rough surface,partially detached from glass wall rADAMTS016-5 porous lyocake, roughsurface, partially detached from glass wall rADAMTS016-6 no lyocake,only airy film rADAMTS016-7 compact, porous lyocake, rough surface,partially detached from glass wall rADAMTS016-8 no lyocake, only airyfilm rADAMTS016-9 airy, smooth film rADAMTS016-10 powdery, smoothsurface rADAMTS016-11 no lyocake, only airy film rADAMTS016-12 powdery,smooth surface

Z. Example 26 Long Term Temperature Stress Test

To further characterize the stability of lyophilized formulations ofADAMTS13, a second long term stress test was initiated. For this test,recombinant human ADAMTS13 was formulated at three final proteinconcentrations according to Table 19. A second sugar (mannitol) wasincluded in the formulation as it was found to stabilize the proteinduring lyophilization and provided a compact lyocake with a smoothsurface. All stress test samples were characterized by DLS (FIG. 39),SE-HPLC (Table 20), and FTIR. FRETS activity (Table 21) and A13 antigenELISA (Table 22) were also measured over time for all of theformulations.

TABLE 19 His-Buffer rADAMTS13 formulation for stress test (rADAMTS025)rADAMTS025-1 rADAMTS025-2 rADAMTS025-3 Concentration 600 300 150 [UFRETS/mL] NaCl [mM] 30 30 30 Histidine [mM] 20 20 20 CaCl₂ [mM] 2 2 2Polysorbate 80 0.05 0.05 0.05 [%] Sucrose [%] 1 1 1 Mannitol [%] 3 3 3pH 7.0 7.0 7.0

TABLE 20 Influence of high ADAMTS13 concentration on the aggregate levelas determined by SE-HPLC (rADAMTS025). Dimer/ Aggregate Oligomer MonomerLot [%] [%] [%] rADAMTS025-1 start 0.15 4.40 95.45 rADAMTS025-1 3 months+4° C. 0.29 4.60 95.10 rADAMTS025-1 2 months 30° C. 0.36 4.44 95.20rADAMTS025-1 3 months 30° C. 0.31 5.22 94.47 rADAMTS025-1 1 months 40°C. 0.19 5.30 94.51 rADAMTS025-1 2 months 40° C. 0.23 5.34 94.43rADAMTS025-1 3 months 40° C. 0.43 5.67 93.90

TABLE 21 FRETS-VWF73 activity in lyophilized ADAMTS13 formulations.[U/ml] [U/ml] [U/ml] Prefor- Before Before [U/ml] [%] [U/ml] [%] [U/ml][%] [U/ml] [%] [U/ml] [%] [U/ml] [%] mulation Filtration LYO 0 M 1 M 2 M3 M 4 M 6 M -1 A  4° C. 685.6 559 528 522 100% — — — — 482.20 92% 462.4089% 411.80 79% 30° C. — — 441 84% 471.20 90% 458.30 88% 386.70 74% 40°C. 475 91% 402 77% 441.80 85% 416.30 80% 367.50 70% -2 A  4° C. 278 258252 100% — — — — 236.6 94% 229.9 91% 210.7 84% 30° C. — — 227 90% 231.692% 220.7 88% 198.2 79% 40° C. 243 96% 209 83% 212 84% 205.8 82% 181.272% -3 A  4° C. 129 122 117 100% — — — — 115 98% 112.4 98% 107.1 92% 30°C. — — 106 91% 114 97% 103.1 88% 98.2 84% 40° C. 111 95%  98 84% 99.185% 95.5 82% 84.4 72%

TABLE 22 Recovery of A13 antigen in lyophilized ADAMTS13 formulations.[μg/ml] [μg/ml] [μg/ml] Prefor- Before Before [μg/ml] [%] [μg/ml] [%][μg/ml] [%] [μg/ml] [%] [μg/ml] [%] mulation Filtration LYO 0 M 1 M 2 M3 M 4 M -1 A  4° C. 1044.898 602 590 586 100% — — — — 671 114% 588 100%30° C. — — 600 102% 689 118% 582  99% 40° C. 574 98% 630 107% 682 116%562  96% -2 A  4° C. 324 339 321 100% — — — — 355 111% 344 107% 30° C. —— 348 108% 323 101% 336 105% 40° C. 321 100% 347 108% 351 110% 353 110%-3 A  4° C. 150 152 127 100% — — — — 166 131% 162 128% 30° C. — — 169133% 177 139% 166 131% 40° C. 147 116% 165 130% 176 139% 168 133%

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1.-20. (canceled)
 21. A method of treating or preventing a thromboticdisease or condition, the method comprising administering to a subjectin need thereof a therapeutically effective dose of an ADAMTS13formulation, wherein said formulation comprises: (a) 0.05 mg/ml to 10.0mg/ml ADAMTS13; (b) 0 mM to 100 mM of a pharmaceutically acceptable salt(c) 0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; and (f) a buffering agent for maintaining a pHbetween 6.0 and 8.0.
 22. The method of treating or preventing of claim21, wherein said thrombotic disease or condition is selected from thegroup consisting of acquired thrombotic thrombocytopenic purpura (TTP),hereditary thrombotic thrombocytopenic purpura (TTP) thromboticmicroangiopathy, thrombocytopenia, microvascular thrombosis, arterialthrombosis, acute myocardial infarction (AMI), stroke, sepsis, anddisseminated intravascular coagulation (DIC).
 23. The method of treatingor preventing of claim 21, wherein said formulation comprises betweenabout 50 units and about 1000 units of ADAMTS13 activity per mL.
 24. Themethod of treating or preventing of claim 21, wherein saidpharmaceutically acceptable salt is sodium chloride (NaCl).
 25. Themethod of treating or preventing of claim 21, wherein said formulationcomprises between 1.0 and 10.0 mM calcium.
 26. The method of treating orpreventing of claim 21, wherein said formulation comprises between 2%and 6% of a sugar and/or sugar alcohol.
 27. The method of treating orpreventing of claim 21, wherein said sugar and/or sugar alcohol isselected from the group consisting of sucrose, trehalose, mannitol, anda combination thereof.
 28. The method of treating or preventing of claim21, wherein said sugar and/or sugar alcohol is a combination of sucroseand mannitol.
 29. The method of treating or preventing of claim 21,wherein said formulation comprises between 0.01% and 0.1% of a non-ionicsurfactant.
 30. The method of treating or preventing of claim 21,wherein said surfactant is selected from the group consisting ofPolysorbate 20, Polysorbate 80, Pluronic F-68, and BRIJ
 35. 31. Themethod of treating or preventing of claim 21, wherein said formulationcomprises between 5 mM and 100 mM of a buffering agent.
 32. The methodof treating or preventing of claim 21, wherein said formulationcomprises between 10 mM and 50 mM of a buffering agent.
 33. The methodof treating or preventing of claim 21, wherein said buffering agent ishistidine or HEPES.
 34. The method of treating or preventing of claim21, wherein said formulation has a pH between 6.5 and 7.5.
 35. Themethod of treating or preventing of claim 21, wherein said pH of theformulation is 7.0±0.2.
 36. The method of treating or preventing ofclaim 21, wherein said formulation further comprises between 0.5 μM and20 μM zinc.
 37. The method of treating or preventing of claim 21,wherein said formulation comprises between 0 mM and 90 mM of apharmaceutically acceptable salt.
 38. The method of treating orpreventing of claim 21, wherein said formulation comprises between 0 mMand 60 mM of a pharmaceutically acceptable salt.
 39. The method oftreating or preventing of claim 21, wherein said formulation comprisesbetween 0 mM and 30 mM of a pharmaceutically acceptable salt.
 40. Themethod of treating or preventing of claim 21, wherein said formulationcomprises between 30 mM and 60 mM of a pharmaceutically acceptable salt.41. The method of treating or preventing of claim 21, wherein saidformulation comprises between 30 mM and 90 mM of a pharmaceuticallyacceptable salt.
 42. The method of treating or preventing of claim 21,wherein said formulation comprises between 60 mM and 90 mM of apharmaceutically acceptable salt.
 43. The method of treating orpreventing of claim 21, wherein said formulation comprises monomericADAMTS13 protein and a content of ADAMTS13 aggregates of less than 5%total protein.
 44. The method of treating or preventing of claim 21,wherein the specific activity of the ADAMTS13 protein is at least about600 U of FRETS-VWF73 activity per mg ADAMTS13 protein.
 45. The method oftreating or preventing of claim 21, wherein said formulation comprises:(a) 0.05 mg/ml to 10.0 mg/ml ADAMTS13; (b) 0 mM to 60 mM NaCl; (c) 2 mMto 4 mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2% sucrose; (f)0.025% to 0.1% Polysorbate 80; (g) 10 mM to 50 mM histidine; and (h) apH of 7.0.+−.0.2.
 46. The method of treating or preventing of claim 21,wherein said formulation comprises: (a) 0.05 mg/ml to 10.0 mg/mlADAMTS13; (b) 0 mM to 100 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) a sugar and/or sugar alcohol; (e) anonionic surfactant; (f) 5 mM to 100 mM of a buffering agent selectedfrom the group consisting of histidine and HEPES; and (g) a pH between6.0 and 8.0.
 47. The method of treating or preventing of claim 21,wherein said formulation comprises: (a) 0.05 mg/ml to 10.0 mg/mlADAMTS13; (b) 0 mM to 100 mM of a pharmaceutically acceptable salt; (c)0.5 mM to 20 mM calcium; (d) 2% to 6% of a sugar and/or sugar alcohol;(e) a nonionic surfactant; (f) 5 mM to 100 mM of a buffering agentselected from the group consisting of histidine and HEPES; and (g) a pHbetween 6.0 and 8.0.
 48. A method of treating or preventing aninfarction or condition associated with an infarction, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective dose of an ADAMTS13 formulation, wherein said formulationcomprises: (a) 0.05 mg/ml to 10.0 mg/ml ADAMTS13; (b) 0 mM to 100 mM ofa pharmaceutically acceptable salt (c) 0.5 mM to 20 mM calcium; (d) asugar and/or sugar alcohol; (e) a nonionic surfactant; and (f) abuffering agent for maintaining a pH between 6.0 and 8.0.
 49. The methodof treating or preventing of claim 48, wherein said infarction orcondition associated with said infarction is selected from the groupconsisting of myocardial infarction, pulmonary embolism, cerebrovascularevents such as stroke, peripheral artery occlusive disease,antiphospholipid syndrome, sepsis, giant-cell arteritis (GCA), hernia,and volvulus.
 50. The method of treating or preventing of claim 48,wherein said formulation comprises between about 50 units and about 1000units of ADAMTS13 activity per mL.
 51. The method of treating orpreventing of claim 48, wherein said pharmaceutically acceptable salt issodium chloride (NaCl).
 52. The method of treating or preventing ofclaim 48, wherein said formulation comprises between 1.0 and 10.0 mMcalcium.
 53. The method of treating or preventing of claim 48, whereinsaid formulation comprises between 2% and 6% of a sugar and/or sugaralcohol.
 54. The method of treating or preventing of claim 48, whereinsaid sugar and/or sugar alcohol is selected from the group consisting ofsucrose, trehalose, mannitol, and a combination thereof.
 55. The methodof treating or preventing of claim 48, wherein said sugar and/or sugaralcohol is a combination of sucrose and mannitol.
 56. The method oftreating or preventing of claim 48, wherein said formulation comprisesbetween 0.01% and 0.1% of a non-ionic surfactant.
 57. The method oftreating or preventing of claim 48, wherein said surfactant is selectedfrom the group consisting of Polysorbate 20, Polysorbate 80, PluronicF-68, and BRIJ
 35. 58. The method of treating or preventing of claim 48,wherein said formulation comprises between 5 mM and 100 mM of abuffering agent.
 59. The method of treating or preventing of claim 48,wherein said formulation comprises between 10 mM and 50 mM of abuffering agent.
 60. The method of treating or preventing of claim 48,wherein said buffering agent is histidine or HEPES.
 61. The method oftreating or preventing of claim 48, wherein said formulation has a pHbetween 6.5 and 7.5.
 62. The method of treating or preventing of claim48, wherein said pH of the formulation is 7.0±0.2.
 63. The method oftreating or preventing of claim 48, wherein said formulation furthercomprises between 0.5 μM and 20 μM zinc.
 64. The method of treating orpreventing of claim 48, wherein said formulation comprises between 0 mMand 90 mM of a pharmaceutically acceptable salt.
 65. The method oftreating or preventing of claim 48, wherein said formulation comprisesbetween 0 mM and 60 mM of a pharmaceutically acceptable salt.
 66. Themethod of treating or preventing of claim 48, wherein said formulationcomprises between 0 mM and 30 mM of a pharmaceutically acceptable salt.67. The method of treating or preventing of claim 48, wherein saidformulation comprises between 30 mM and 60 mM of a pharmaceuticallyacceptable salt.
 68. The method of treating or preventing of claim 48,wherein said formulation comprises between 30 mM and 90 mM of apharmaceutically acceptable salt.
 69. The method of treating orpreventing of claim 48, wherein said formulation comprises between 60 mMand 90 mM of a pharmaceutically acceptable salt.
 70. The method oftreating or preventing of claim 48, wherein said formulation comprisesmonomeric ADAMTS13 protein and a content of ADAMTS13 aggregates of lessthan 5% total protein.
 71. The method of treating or preventing of claim48, wherein the specific activity of the ADAMTS13 protein is at leastabout 600 U of FRETS-VWF73 activity per mg ADAMTS13 protein.
 72. Themethod of treating or preventing of claim 48, wherein said formulationcomprises: (a) 0.05 mg/ml to 10.0 mg/ml ADAMTS13; (b) 0 mM to 60 mMNaCl; (c) 2 mM to 4 mM calcium; (d) 2% to 4% mannitol; (e) 0.5% to 2%sucrose; (f) 0.025% to 0.1% Polysorbate 80; (g) 10 mM to 50 mMhistidine; and (h) a pH of 7.0.+−.0.2.
 73. The method of treating orpreventing of claim 48, wherein said formulation comprises: (a) 0.05mg/ml to 10.0 mg/ml ADAMTS13; (b) 0 mM to 100 mM of a pharmaceuticallyacceptable salt; (c) 0.5 mM to 20 mM calcium; (d) a sugar and/or sugaralcohol; (e) a nonionic surfactant; (f) 5 mM to 100 mM of a bufferingagent selected from the group consisting of histidine and HEPES; and (g)a pH between 6.0 and 8.0.
 74. The method of treating or preventing ofclaim 48, wherein said formulation comprises: (a) 0.05 mg/ml to 10.0mg/ml ADAMTS13; (b) 0 mM to 100 mM of a pharmaceutically acceptablesalt; (c) 0.5 mM to 20 mM calcium; (d) 2% to 6% of a sugar and/or sugaralcohol; (e) a nonionic surfactant; (f) 5 mM to 100 mM of a bufferingagent selected from the group consisting of histidine and HEPES; and (g)a pH between 6.0 and 8.0.